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| United States Patent |
6,011,001
|
|
Navia, ;, , , -->
Navia
, et al.
|
January 4, 2000
|
Method of protein therapy by orally administering crosslinked protein
crystals
Abstract
A protein such as an enzyme or antibody is immobilized by crosslinking
crystals of the protein with a multifunctional crosslinking agent. The
crosslinked protein crystals may be lyophilized for storage. A preferred
protein is an enzyme such as thermolysin, elastase, asparaginase,
lysozyme, lipase or urease. Crosslinked enzyme crystals preferably retain
at least 91% activity after incubation for three hours in the presence of
a concentration of Pronase.TM. that causes the soluble uncrosslinked form
of the enzyme to lose at least 94% of its initial activity under the same
conditions. A preferred enzyme:Pronase.TM. ratio is 40:1. Enzyme crystals
that are crosslinked may be microcrystals having a cross-section of
10.sup.-1 mm or less. Crosslinked enzyme or antibody crystals may be used
in an assay, diagnostic kit or biosensor for detecting an analyte, in an
extracorporeal device for altering a component of a fluid, in producing a
product such as using crosslinked thermolysin crystals to produce
aspartame and in separating a substance from a mixture. Enzyme or
non-enzyme protein therapy can be performed by administering orally
crosslinked enzyme crystals or crosslinked non-enzyme protein crystals
that have a therapeutic affect. The crosslinked crystals have improved
stability to proteases in the gut. Crosslinked lipase crystals may be
administered for treatment where there is pancreatic insufficiency and/or
fat malabsorption conditions in which lipase secretion is abnormally low.
| Inventors:
|
Navia; Manuel A. (Lexington, MA);
St. Clair; Nancy L. (Charlestown, MA)
|
| Assignee:
|
Vertex Pharmaceuticals, Inc. (Cambridge, MA)
|
| Appl. No.:
|
484978 |
| Filed:
|
June 7, 1995 |
| Current U.S. Class: |
514/2; 424/94.1; 424/94.6; 424/94.63; 435/41; 435/109; 435/174; 435/195; 435/198; 435/212; 435/218; 435/817; 436/518; 530/402; 530/413; 530/810 |
| Intern'l Class: |
A61K 038/00; A61K 038/43; A61K 039/00; C07K 001/00 |
| Field of Search: |
435/41,174,176,177,180,816,109,195,198,212,218,817
514/2
436/518
530/402,413,810
424/94.1,94.6,94.63
|
References Cited
U.S. Patent Documents
| Re33441 | Nov., 1990 | Wumpelman et al. | 435/94.
|
| 4373023 | Feb., 1983 | Langer et al. | 435/2.
|
| 4390632 | Jun., 1983 | Carter | 435/183.
|
| 4411996 | Oct., 1983 | Lloyd | 435/94.
|
| 4693985 | Sep., 1987 | Degen et al. | 436/531.
|
| 4699882 | Oct., 1987 | Visuri | 435/188.
|
| 4892825 | Jan., 1990 | Wumpelmann et al. | 435/94.
|
| 5120650 | Jun., 1992 | Visuri | 435/176.
|
| Foreign Patent Documents |
| 0 092 829 | Nov., 1983 | EP.
| |
| 0 169 767 | Jan., 1986 | EP.
| |
| 0 166 427 | Jan., 1986 | EP.
| |
| 0 175 582 | Mar., 1986 | EP.
| |
| 0 195 311 | Sep., 1986 | EP.
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| 0341503 | Nov., 1989 | EP.
| |
| 0 341 503 | Nov., 1989 | EP.
| |
| 0 367 302 | May., 1990 | EP.
| |
| WO 85/03247 | Aug., 1985 | WO.
| |
| WO 91/05857 | May., 1991 | WO.
| |
Other References
Derwent Abstract 88-06122 of Japanese patent application 63017691,
published Sep. 1988.
G. M. Alter et al., "Kinetic Properties of Carboxypeptidase B in Solutions
and Crystals", Biochemistry, 16, pp. 3663-3668 (1977).
S.A. Barker et al., "Enzymatic Processes for High-Fructose Corn Syrup," In:
Enzymes and Immobilized Cells in Biotechnology, Menlo Park, CA (1985).
G.J. Bartling et al., "Protein Modification in Nonaqueous Media -A New
Method of Enzyme Cross-Linking", Enzyme, 18, pp. 310-316 (1974).
S.J. Bayne et al., "Enzymatically Active, Cross-Linked Pig Heart Lactate
Dehydrogenase Crystals", Carlsberg Res. Comm., 41, pp. 221-216 (1976).
W.H. Bishop et al., "Isoelectric Point of a protein in the Crosslinked
Crystalline State: .beta.-Lactoglobulin", J. Mol. Biol., 33, pp. 415-421
(1968).
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Cross-Linked Carboxypeptidase A", Thermochimica Acta, 8, pp. 455-464
(1974).
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Lysozyme Crystals", Biophys. J., 8, pp. 549-455 (1968).
H.C. Hedrich et al., "Large-Scale Purification, Enzymic Characterization,
and Crystallization of the Lipase from Geotrichum Candidum", Enzyme &
Microb. Technol., 13, pp. 840-847 (1991).
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P.J. Kasvinsky et al., "Activity of Glycogen Phosphorylase in the
Crystalline State ", J. Biol. Chem., 251, pp. 6852-6859 (1976).
H. Kirsten et al., "Catalytic Activity of Non-Cross-Linked Microcrystals of
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pp. 427-434 (1983).
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in Biochem. Sci., 14, pp. 141-144 (1989).
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1062-1064 (1987).
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Corn Syrup and Other Corn Sweetners" (publication date unknown).
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pp. 474-476 (1984).
T. Nakagawa et al., "Development of Effective Cross-Linking Method for
Bioactive Substance-Enzyme Immobilization Using Glutaraldehyde Oligomers",
Chem. Pharm. Bull., 37, pp. 2463-2466 (1989).
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Peroxidases", In: Enzymes in Biomass Conversion, Am. Chem. Soc., Boston,
MA, Apr. 22-27, (1990).
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Crystalline State: Carboxypeptidase-A", Proc. Nat. Acad. Sci., 51, pp.
833-839 (1964).
F.A. Quiocho et al., "Effects of Changes in some Solvent Parameters on
Carboxypeptidase A in Solution and in Cross-Linked Crystals", Proc. Nat.
Acad. Sci., 57, pp. 525-537 (1967).
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Active Site of Elastase", Cold Spring Harbour Symp. Quant. Bio., XXXVI,
pp. 91-105 (1972).
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Carboxypeptidase A", Biochemistry, 16, pp. 1142-1150 (1977).
T. Tashima et al., "Structure of a New Oligomer of Glutaraldehyde Produced
by Aldol Condensation Reaction", J. Org. Chem., 56, pp. 694-697 (1991).
V.P. Torchlin et al., "The Principles of Enzyme Stabilization: III. The
Effects of the Length of Intra-Molecular Cross-Linkages on the
Thermostability of Enzymes", Biochem. Biophys. Acta, 522, pp. 277-283
(1978).
V.P. Torchlin et al., "Principles of Enzyme Stabilization: V. The
Possibility of Enzyme Selfstabilization Under the Action of Potentially
Reversible Intramolecular Cross-Linkages of Different Length", Biochem.
Biophys. Acta, 568, pp. 1-10 (1979).
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Crystalline State", Carlsberg Res. Comm., 42, pp. 407-420 (1977).
K. Visuri et al., "Purification and Characterisation of Crystalline
.beta.-Amylase From Barley", Eur. J. Biochem., 28, pp. 555-565 (1972).
K. Visuri et al., "Enzymatic Production of High Fructose Corn Syrup (HFCS)
Containing 55% Fructose in Aqueous Ethanol", Biotech. & Bioeng., 30, pp.
917-920 (1987).
K. Visuri, "Industrial Scale Crystallization of Glucose Isomerase", Enzyme
Engineering X: International Conference, Sep. 24-29, 1989, Kashikojima,
Japan.
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Kozlony es Vedjegyertesito, Budapest, 1990. Abstract T/52 817 (Original in
Hungarian, English translation).
K. Visuri, "Crosslinked Crystalline Glucose Isomerase As Industrial
Catalyst", Lecture in Detmold Starch Convention (1992).
A. Yonath et al., "Crystallographic Studies of Protein Denaturation and
Renaturation. 1. Effects of Denaturants on Volume and X-ray Pattern of
Cross-Linked Triclinic Lysozyme Crystals", Biochemistry, 16, pp. 1413-1417
(1977).
Product Information SPEZYME.sup.R CIGI, Genecor International, (publication
date unknown).
K.M. Lee et al., "Crosslinked Cystalline Horse Liver Alcohol Dehydrogenase
As a Redox Catalyst: Activity and Stability Toward Organic Solvent",
Bioorganic Chem., 14, pp. 202-210 (1986).
Lee, et al., Bioorganic Chemistry, vol. 14, 1986, pp. 202-210.
Nakanishi, et al., Bio/Technology, vol. 3, 1985, pp. 459-464.
|
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: Fish & Neave, Haley, Jr.; James F., Pierri; Margaret A.
Parent Case Text
This application is a continuation of application Ser. No. 08/017,510,
filed Feb. 12, 1993, now U.S. Pat. No. 5,618,710, which is a
continuation-in-part of application Ser. No. 07/864,424, filed Apr. 6,
1992, now abandoned, which is a continuation-in-part of application Ser.
No. 07/720,237, filed Jun. 24, 1991, now abandoned, which is a
continuation-in-part of application Ser. No. 07/562,280, filed Aug. 3,
1990, now abandoned. The teachings of application Ser. Nos. 07/864,424,
07/720,237 and 07/562,280 are incorporated herein by reference.
Claims
We claim:
1. A method for carrying out protein therapy in an individual, comprising
the step of administering orally to said individual a protein crystal
crosslinked with a multifunctional crosslinking agent, said crosslinked
protein crystal having resistance to exogenous proteolysis, such that said
crosslinked protein crystal retains at least 91% of its stability, as
measured in terms of degradation, after incubation for three hours in the
presence of a concentration of Pronase.TM. that causes the soluble
uncrosslinked form of the protein that is crystallized to form said
protein crystal that is crosslinked to lose at least 94% of its stability,
as measured in terms of degradation, under the same conditions.
2. The method according to claim 1, wherein said crystal is a microcrystal.
3. The method according to claim 2, wherein said microcrystal has a
cross-section of 10.sup.-1 mm or less.
4. The method according to claim 1, wherein said crystal is in lyophilized
form.
Description
BACKGROUND OF THE INVENTION
Enzymes are used as industrial catalysts for the large and laboratory scale
economical production of fine and specialty chemicals (Jones, J. B.,
Tetrahedron 42: 3351-3403 (1986)), for the production of foodstuffs (Zaks
et. al., Trends in Biotechnology 6: 272-275 (1988)), and as tools for the
synthesis of organic compounds (Wong, C. -H., Science 244: 1145-1152
(1989); CHEMTRACTS-Org. Chem. 3: 91-111 (1990); Klibanov, A. M., Acc.
Chem. Res. 23: 114-120 (1990)).
Enzyme-based manufacturing can significantly reduce the environmental
pollution burden implicit in the large scale manufacturing of otherwise
unusable chemical intermediates, as shown in the large scale production of
acrylamide using the enzyme, nitrile hydratase (Nagasawa, T. and Yamada,
H., Trends in Biotechnology 7: 153-158 (1989)).
Enzymes are also used in biosensor applications to detect various
substances of clinical, industrial and other interest (Hall, E.,
"Biosensors", Open University Press (1990)). In the clinical area, enzymes
may be used in extracorporeal therapy, such as hemodialysis and
hemofiltration, where the enzymes selectively remove waste and toxic
materials from blood (Klein, M. and Langer, R., Trends in Biotechnology 4:
179-185 (1986)). Enzymes are used in these areas because they function
efficiently as catalysts for a broad range of reaction types, at modest
temperatures, and with substrate specificity and stereoselectivity.
Nonetheless, there are disadvantages associated with the use of soluble
enzyme catalysts which have limited their use in industrial and laboratory
chemical processes (Akiyama et. al., CHEMTECH 627-634 (1988)).
Enzymes are expensive and relatively unstable compared to most industrial
and laboratory catalysts, even when they are used in aqueous media where
enzymes normally function. Many of the more economically interesting
chemical reactions carried out in common practice are incompatible with
aqueous media, where, for example, substrates and products are often
insoluble or unstable, and where hydrolysis can compete significantly. In
addition, the recovery of soluble enzyme catalyst from product and
unreacted substrate in the feedstock often requires the application of
complicated and expensive separation technology. Finally, enzymes are
difficult to store in a manner that retains their activity and functional
integrity, for commercially reasonable periods of time (months to years)
without having to resort to refrigeration (4.degree. C. to -80.degree. C.
to liquid N.sub.2 temperatures), or to maintenance in aqueous solvents of
suitable ionic strength, pH, etc.
Enzyme immobilization methods have, in many instances, circumvented these
disadvantages. Immobilization can improve the stability of enzyme
catalysts and protect their functional integrity in the harsh solvent
environments and extreme temperatures characteristic of industrial and
laboratory chemical processes (Hartmeier, W., Trends in Biotechnology 3:
149-153 (1985)). Continuous flow processes may be operated with
immobilized enzyme particles in columns, for example, where the soluble
feedstock passes over the particles and is gradually converted into
product. As used herein, the term enzyme immobilization refers to the
insolubilization of enzyme catalyst by attachment to, encapsulation of, or
by aggregation into macroscopic (10.sup.-1 mm) particles.
A number of useful reviews of enzyme immobilization methods have appeared
in the literature (Maugh, T. H., Science 223: 474-476 (1984); Tramper, J.,
Trends in Biotechnology 3: 45-50 (1985)). Maugh describes five general
approaches to the immobilization of enzymes. These include: adsorption on
solid supports (such as ion-exchange resins); covalent attachments to
supports (such as ion-exchange resins, porous ceramics or glass beads);
entrapment in polymeric gels; encapsulation; and the precipitation of
soluble proteins by cross-linking them with bifunctional reagents in a
random and undefined manner. In addition, one can immobilize whole cells
(usually dead and made permeable) which have expressed the desired enzyme
activity at high levels (e.g., Nagasawa, T. and Yamada, H., Trends in
Biotechnology 7: 153-158 (1989)).
Each of these immobilization procedures has its own advantages and
limitations and none can be considered optimal or dominating. In most of
them, the enzyme catalyst ultimately represents only a small fraction of
the total volume of material present in the chemical reactor. As such, the
bulk of the immobilized medium is made up of inert, but often costly
carrier material. In all of them, the immobilizing interactions of the
enzyme catalyst molecules with each other and/or with the carrier material
tend to be random and undefined. As a result, although these interactions
confer some enhanced stability to the enzyme catalyst molecules, their
relative non-specificity and irregularity makes that stabilization
sub-optimal and irregular. In most cases, access to the active site of the
enzyme catalyst remains ill-defined. In addition, the immobilization
methods described above fail to deal with problems associated with storage
and refrigeration. Nor can conventionally immobilized enzymes generally be
manipulated, as in being exchanged into one or another solvent of choice,
without risk to the structural and functional integrity of the enzyme. In
practical terms, except for the attached tether to the carrier particle,
conventionally immobilized enzymes bear close resemblance to soluble
enzymes, and share with them a susceptibility to denaturation and loss of
function in harsh environments. In general, immobilization methods lead to
a reduction of observed enzyme-catalyzed reaction rates relative to those
obtained in solution. This is mostly a consequence of the limits of inward
diffusion of substrate and outward diffusion of product within the
immobilized enzyme particle (Quiocho, F. A., and Richards, F. M.,
Biochemistry 5: 4062-4076 (1967)). The necessary presence of inert carrier
in the immobilized enzyme particles increases the mean free path between
the solvent exterior of the immobilized enzyme particle and the active
site of the enzyme catalyst and thus exacerbates these diffusion problems.
When dealing with immobilized cells, the diffusion problem is particularly
severe, even if cell walls and membranes are made permeable to substrate
and product in some way. One would further be concerned with the multitude
of contaminating enzymatic activities, metabolites, and toxins contained
in cells, and with the stability of cells in harsh solvents or extreme
temperature operating environments. An improved immobilization technique
which avoids the limitations of the presently available methods would be
helpful in promoting the use of enzymes as industrial catalysts,
particularly if it were shown to be useful on a large scale (Daniels, M.
J., Methods in Enzymology 1.36: 371-379 (1987)).
SUMMARY OF THE INVENTION
The present invention relates to a method of immobilizing a protein,
particularly an enzyme or an antibody, by forming crystals of the enzyme
or antibody and, generally, also crosslinking the resulting crystals
through use of a bifunctional reagent; crosslinked immobilized enzyme
crystals (referred to as CLECs or CLIECs) made by this method; crosslinked
immobilized antibody crystals (referred to as CLACs); the lyophilization
of the resulting crystals as a means of improving the storage, handling,
and manipulation properties of immobilized enzymes and antibodies; a
method of making a desired product by means of a reaction catalyzed by a
CLEC or a set of CLECs; and methods in which the CLACs of the present
invention are used, such as a method of separating or purifying a
substance or molecule of interest, in which a CLAC which recognizes
(binds) the substance or molecule of interest serves as an immunospecific
reagent. In another embodiment, a CLAC can be used for detection of a
substance or molecule of interest in a sample, such as a biological
sample, water, or other sample; this embodiment is useful, for example,
for diagnostic purposes. In a further embodiment, CLACs of the present
invention can be used for therapeutic purposes, in much the same manner
monoclonal antibodies are now used therapeutically; in many instances, a
CLAC of a particular enzyme can simply replace or substitute for
presently-used (non-CLAC) antibodies. A particular advantage to CLACs is
their enhanced resistance to degradation (e.g., enhanced protease
resistance), relative to that of non-CLAC antibodies.
In the method of the present invention by which enzyme crystals are
produced, small protein crystals (crystals of approximately 10.sup.-1 mm
in size) are grown from aqueous solutions, or aqueous solutions containing
organic solvents, in which the enzyme catalyst is structurally and
functionally stable. In a preferred embodiment, crystals are then
crosslinked with a bifunctional reagent, such as glutaraldehyde. This
crosslinking results in the stabilization of the crystal lattice contacts
between the individual enzyme catalyst molecules constituting the crystal.
As a result of this added stabilization, the crosslinked immobilized
enzyme crystals can function at elevated temperatures, extremes of pH and
in harsh aqueous, organic, or near-anhydrous media, including mixtures of
these. That is, a CLEC of the present invention can function in
environments incompatible with the functional integrity of the
corresponding uncrystallized, uncrosslinked, native enzyme or
conventionally immobilized enzyme catalysts. CLACs can be made in a
similar manner, using commercially available antibodies or antibodies
produced against a specific antigen or hapten; entire antibodies or
antibody fragments (e.g., FAb fragments) can be used to produce a
corresponding CLAC.
In addition, CLECs made by this method can be subjected to lyophilization,
producing a lyophilized CLEC which can be stored in this lyophilized form
at non-refrigerated (room) temperatures for extended periods of time, and
which can be easily reconstituted in aqueous, organic, or mixed
aqueous-organic solvents of choice, without the formation of amorphous
suspensions and with minimal risk of denaturation.
The present invention also relates to CLECs produced by the present method
and to their use in laboratory and large scale industrial production of
selected materials, such as chiral organic molecules, peptides,
carbohydrates, lipids, or other chemical species. Presently, these are
typically prepared by conventional chemical methods, which may require
harsh conditions (e.g. aqueous, organic or near-anhydrous solvents, mixed
aqueous/organic solvents or elevated temperatures) that are incompatible
with the functional integrity of uncrystallized, uncrosslinked, native
enzyme catalyst. Other macromolecules with catalytic activity can also be
incorporated into the proposed CLEC technology. These might include
catalytic antibodies (Lerner, R. A., Benkovic, S. J., and Schultz, P. G.,
Science 252:659-667 (1991)) and catalytic polynucleotides (Cech, T. R.,
Cell 64:667-669 (1991); Celander, D. W., and Cech, T. R. Science,
251:401-407 (1991)).
The present invention also relates to a method of making a selected product
by means of a reaction catalyzed by a CLEC of the present invention.
In an example of the method and practice of the present invention, the
enzyme thermolysin, a zinc metalloprotease, was used to synthesize a
chiral precursor of the dipeptidyl artificial sweetener, aspartame.
(Example 1) The enzyme thermolysin was crystallized from a starting
aqueous solution of 45% dimethyl sulfoxide, and 55% 1.4M calcium acetate,
0.05M sodium cacodylate, pH 6.5. The resulting crystals were cross-linked
with glutaraldehyde to form a thermolysin CLEC. The thermolysin CLEC was
then transferred from the aqueous crystallization solution in which it was
made, into a solution of ethyl acetate containing the substrates,
N-(benzyloxycarbonyl)-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl
ester (L-Phe-OMe). The thermolysin CLEC was then used to catalyze a
condensation reaction of the two substrates to synthesize
N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester
(Z-L-Asp-L-Phe-OMe), which is the dipeptidyl precursor of the artificial
sweetener aspartame. Using any one of many known techniques (see, e.g.
Lindeberg, G., J. Chem Ed. 64: 1062-1064 (1987)) the L-aspartic acid in
the synthesized dipeptidyl precursor can be deprotected by the removal of
the benzyloxycarbonyl (Z-) group to produce aspartame (L-Asp-L-Phe-OMe).
In a second example of the method and practice of the present invention,
the enzyme thermolysin was used to produce thermolysin CLECs. The activity
and stability of thermolysin CLECs were compared to that of soluble
thermolysin under optimum conditions and conditions of extreme pH and
temperature, following incubation in the presence of organic solvents and
following incubation in the presence of exogenous protease. (Example 2)
See also Example 3, which describes further work with thermolysin CLEC.
The enzyme thermolysin was crystallized from a solution of 1.2M calcium
acetate and 30% dimethyl sulfoxide pH 8.0. The resulting crystals were
crosslinked with glutaraldehyde at a concentration of 12.5% to form a
thermolysin CLEC. The thermolysin CLEC was then lyophilized by a standard
procedure (Cooper, T. G., The Tools of Biochemistry, pp. 379-380 (John
Wiley and Sons, N.Y. (1977)) to form a lyophilized enzyme CLEC of
thermolysin. This lyophilized CLEC was then transformed directly into the
different aqueous, organic, and mixed aqueous/organic solvents of choice
without an intervening solvent exchange procedure, without formation of
amorphous suspensions, and with minimal risk of denaturation. These
solvents included acetonitrile, dioxane, acetone, and tetrahydrofuran, but
not to the exclusion of others. Following incubation, activity was assayed
spectrophotometrically by cleavage of the dipeptide substrate FAGLA
(furylacryloyl-glycyl-L-leucine amide).
In a third example of the method and practice of the present invention, the
enzyme elastase (porcine pancreatic) was crystallized from an aqueous
solution of 5.5 mg/ml protein in 0.1 M sodium acetate at pH 5.0 at room
temperature (Sawyer, L. et al., J. Mol. Biol. 118:137-208). The resulting
crystals were crosslinked with glutaraldehyde at a concentration of 5% to
form an elastase CLEC. (Example 4) The elastase CLEC was lyophilized as
described in Example 2.
In a fourth example of the method and practice of the present invention,
and as disclosed here, the enzyme esterase (porcine liver) was
crystallized from an aqueous solution of 15 mg/ml protein in 0.25 M
calcium acetate at pH 5.6 at room temperature. The resulting crystals were
crosslinked with glutaraldehyde at a concentration of 12.5% to form an
esterase CLEC. (Example 5) The esterase CLEC was lyophilized as described
in Example 2.
In a fifth example of the method and practice of the present invention, and
as disclosed here, the enzyme lipase (Geotrichum candidum) was
crystallized from an aqueous solution of 20 mg/ml protein in 50 mM Tris at
pH 7 at room temperature. The resulting crystals were crosslinked with
glutaraldehyde at a concentration of 12.5% to form a lipase CLEC. The
lipase CLEC was lyophilized as described in Example 2. In addition,
Candida cylindracea lipase has been crystallized and crosslinked, as
described herein; the resulting CLEC was shown to retain significant
enzymatic activity. Further, porcine pancreatic lipase has been
crystallized and cross-linked; preliminary asssessment of the resulting
CLEC showed that it retained less activity than either of the other
lipases (G. candidum or C. cylindracea). (See Examples 6, 10 and 11).
In a sixth example of the method and practice of the present invention, the
enzyme lysozyme (hen egg white) was crystallized from an aqueous solution
of 40 mg/ml protein in 40 mM sodium acetate buffer containing 5% sodium
chloride at pH 7.4 at room temperature (Blake, C. C. F. et al., Nature,
196:1173 (1962)). The resulting crystals were crosslinked with
glutaraldehyde at a concentration of 20% to form a lysozyme CLEC. (Example
7) The lysozyme CLEC was lyophilized as described in Example 2.
In a seventh example of the method and practice of the present invention,
the enzyme asparaginase (Escherichia coli) was crystallized from an
aqueous solution of 25 mg/ml protein in 50 mM sodium acetate and 33%
ethanol at pH 5.0 at 4.degree. C. The crystallization is a modification of
the procedure described by Grabner et al. [U.S. Pat. No. 3,664,926
(1972)]. As disclosed here, the resulting crystals were crosslinked with
glutaraldehyde at a concentration of 7.5% to form an asparaginase CLEC.
(Example 8) The asparaginase CLEC was lyophilized as described in Example
2.
In an eighth example of the method and practice of the present invention,
the enzyme urease (Jack Bean) was crystallized and the resulting urease
crystals were crosslinked. (Example 9) The urease CLEC was lyophilized, as
described in Example 2. Enzymatic activity of the urease CLEC was compared
with that of soluble urease. In addition, enzymatic activity of urease
CLEC in aqueous buffer was compared with activity in sera, a biologically
relevant medium; activity in sera was comparable to activity in aqueous
buffer.
Other enzymes which can be immobilized in a similar manner and used to
catalyze an appropriate reaction include luciferase. Other enzymes, such
as those listed in Tables 1-5, can also be crystallized and crosslinked
using the present method, to produce a desired CLEC which can, in turn, be
used to catalyze a reaction which results in production of a selected
product or to catalyze a reaction which is an intermediate step (i.e. one
in a series of reactions) in the production of a selected product. It is
recognized that although crosslinking helps stabilize the majority of
crystals, it is neither necessary nor desirable in all cases. Some
crystalline enzymes retain functional and structural integrity in harsh
environments even in the absence of crosslinking. Although in the
preferred embodiment, the crystal is crosslinked, crosslinking is not
always necessary to produce an enzyme crystal useful in the present
method.
CLECs have several key characteristics that confer significant advantages
over conventional enzyme immobilization methods presently in use. CLECs
dispense with the need for a separate, inert support structure. Lack of an
inert support will improve substrate and product diffusion properties
within CLECs and provides enzyme concentrations within the crystal that
are close to the theoretical packing limit for molecules of such size.
High enzyme concentrations can lead to significant operational economies
through the increased effective activity of a given volume of catalyst,
reduction in substrate contact time with enzyme and overall reduction in
plant size and capital costs (Daniels, M. J., Methods in Enzymol. 136:
371-379 (1987)). The uniformity across crystal volume and enhanced
stability of the constituent enzyme in CLECs creates novel opportunities
for the use of enzyme catalysis in harsh conditions, such as elevated
temperature, and aqueous, organic or near-anhydrous solvents, as well as
mixtures of these. In addition, the restricted solvent access and regular
protein environment implicit in a crystal lattice should lead to improved
metal ion and co-factor retention for CLECs vs. conventional immobilized
enzyme systems.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphic representation of results of assessment of enzymatic
activity of soluble and thermolysin CLEC.
FIG. 2 is a graphic representation of results of a comparison of pH
dependencies of thermolysin CLEC and soluble thermolysin.
FIG. 3 is a graphic representation of measurement of the activity of
soluble and crystalline thermolysin after incubation at 65.degree. C.
FIG. 4 is a graphic representation of results of assessment of resistance
of soluble and thermolysin CLEC to exogenous proteolytic degradation.
FIGS. 5A-5C show a series of graphic representations of results of
assessment of continuous batch synthesis of the aspartame precursor using
soluble thermolysin and CLEC thermolysin.
FIG. 6 is a graphic representation of results of the assessment of
enzymatic activity for soluble elastase and the corresponding elastase
CLEC.
FIG. 7 is a graphic representation of the resistance of soluble elastase
and the corresponding elastase CLEC to exogenous proteolytic degradation.
FIG. 8 is a graphic representation of results of the assessment of
enzymatic activity for soluble esterase and the corresponding esterase
CLEC.
FIG. 9 is a graphic representation of the resistance of soluble esterase
and the corresponding esterase CLEC to exogenous proteolytic degradation.
FIG. 10 is a graphic representation of results of the assessment of
enzymatic activity for soluble lipase and the corresponding lipase CLEC.
FIG. 11 is a graphic representation of results of the assessment of
enzymatic activity for soluble lysozyme and the corresponding lysozyme
CLEC.
FIG. 12 is a graphic representation of results of the assessment of
enzymatic activity for soluble asparaginase and the corresponding
asparaginase CLEC.
FIG. 13 is a graphic representation of results of the assessment of
enzymatic activity of soluble urease and the corresponding urease CLEC.
FIG. 14 is a graphic representation of results of assessment of pH
dependence and stability of soluble urease and the corresponding urease
CLEC.
FIG. 15 is a graphic representation of results of assessment of thermal
stability of soluble urease and the corresponding urease CLEC.
FIG. 16 is a graphic representation of results of assessment of resistance
to exogenous proteolysis of soluble urease and the corresponding urease
CLEC.
FIG. 17 is a graphic representation of results of assessment of urease CLEC
enzymatic activity in aqueous buffer and in sera.
DETAILED DESCRIPTION OF THE INVENTION
A simple, general procedure that assures stability and function for a given
enzyme or set of enzymes under conditions which are of interest to the
synthetic chemist and which are too harsh for use with enzymes using
presently available methods, would be very useful. Cross-linked
immobilized enzyme crystals (referred to as CLECs or CLIECS) as described
here can be used for this purpose. Stabilization of the crystal lattice
and of the constituent enzyme catalysts in the crystal by the
cross-linking reaction permits the use of CLECs in environments, including
aqueous, organic or near-anhydrous solvents, mixtures of these solvents,
extremes of pH and elevated temperatures, which are incompatible with
enzyme function using presently available methods. In addition, the
stabilization of the crystal lattice in CLECs makes possible the
lyophilization of CLECs by standard methods. Lyophilized CLECs can be
stored for commercially attractive periods of time (months to years) in
the absence of refrigeration, and facilitate the rapid and uncomplicated
utilization of CLECs in industrial and laboratory scale processes by the
simple addition of solvents of choice, without need for intervening
solvent exchange processes. CLECs are also highly resistant to digestion
by exogenous proteases. The method of the present invention facilitates
the use of versatile enzyme catalysts in mainstream industrial chemical
processes, as well as in the laboratory synthesis of novel compounds for
research.
Although crosslinking contributes to the stability of a crystal enzyme, it
is neither necessary nor desirable in all cases. Some crystallized enzymes
retain functional and structural integrity in harsh environments even in
the absence of crosslinking. The preferred embodiment of the present
method includes cross-linking of a crystal enzyme and is described in
detail in the following sections. It is to be understood, however, that
crystallized enzymes which are not subsequently cross-linked can be used
in some embodiments of the present invention.
The regular interactions between the constituent enzyme molecules in the
crystal lattice of a CLEC result in well defined pores of limited size
leading to the enzyme molecules within the body of a CLEC. As a result,
substrates larger than the available pore size will not penetrate the body
of the CLEC particle.
As a consequence of the limited pore size, many enzymatic reactions of
commercial and academic interest involving substrates larger than the pore
size of the CLECs would be beyond the scope of the present invention. This
would include most reactions involving large polymers, such as proteins,
polynucleotides, polysaccharides, and other organic polymers, where the
number of polymeric subunits would be such as to make the polymer larger
than the crystal pore size in CLECs. In such instances, however, catalysis
can still take place on the CLEC surface.
The present invention is a method of immobilizing a selected protein,
particularly an enzyme, by crystallizing and crosslinking the protein,
resulting in production of a crosslinked immobilized enzyme crystal (CLEC)
which can be used to catalyze production of a selected product, such as a
peptide, carbohydrate, lipid or chiral organic molecule. The selected
product can be produced by altering a single substrate (e.g., to produce a
breakdown product or other product) or by combining the substrate with an
additional substance or substances (e.g., a second substrate, molecule or
compound to be added through the action of the CLEC). The present
invention further relates to such CLECs and to a method of making a
selected product by means of a CLEC-catalyzed reaction or CLEC-catalyzed
step in a series of reactions. In one embodiment of the present invention,
the dipeptidyl precursor of aspartame has been produced in a condensation
reaction catalyzed by cross-linked immobilized thermolysin made by the
present method. In another embodiment of this invention, the indicator
substrate, FAGLA, has been cleaved to produce a calorimetric product,
whose presence is indicative of enzyme activity in a thermolysin CLEC.
FAGLA hydrolysis has been used as a model reaction to indicate the
robustness of the thermolysin CLEC in a number of environments that would
be normally incompatible with that enzyme's activity.
In other embodiments of this invention, the enzymes elastase, esterase,
lipase, asparaginase, and lysozyme have been used to cleave various
indicated substances, such as p-nitrophenyl acetate (esterase and lipase),
succinyl-(ala)3-p-nitroanilide (elastase), 4-methylumbelliferyl
N-acetyl-chitrioside (lysozyme) and NADH (asparaginase) and urea (urease).
By the method of this invention, one of ordinary skill in the art can adapt
a protocol for making a desired product by means of a reaction catalyzed
by an immobilized enzyme. The enzyme of interest, when crystallized from
an appropriate solution, can be cross-linked with glutaraldehyde or other
suitable bifunctional reagent in the crystallization solution to produce a
CLEC of that enzyme. Subsequently, the CLEC of the enzyme of choice can be
lyophilized as described in Example 2.
There are several advantages which the use of a CLEC offers over
presently-available enzyme-catalyzed methods. For example, the
cross-linked crystal matrix in a CLEC provides its own support. Expensive
carrier beads, glasses, gels, or films are not required in order to tie
down the enzyme catalyst, as they are in presently-available
immobilization methods. As a result, the concentration of enzyme in a CLEC
is close to the theoretical packing limit that can be achieved for
molecules of a given size, greatly exceeding densities achievable even in
concentrated solutions. The entire CLEC consists of active enzyme (and not
inactive carrier), and thus, the diffusion-related reduction of enzyme
reaction rates usually observed with conventionally immobilized enzymes
relative to enzymes in solution should be minimized, since the mean free
path for substrate and product between active enzyme and free solvent will
be greatly shortened for CLECs (compared to a conventional immobilized
enzyme carrier particles). These high protein densities will be
particularly useful in biosensor, analytical and other applications
requiring large amounts of protein in small volumes. In industrial
processes, the superior performance and compactness of CLECs results in
significant operating economies, by increasing the effective activity of a
given volume of catalyst, thereby allowing reductions in plant size, as
well as capital costs (Daniels, M. J., Methods in Enzymol. 136: 371-379
(1987)). CLECs are relatively monodisperse, with a macroscopic size and
shape reflecting natural crystal growth characteristics of the individual
enzyme catalysts. Replacement of existing carrier-immobilized enzyme media
with CLECs should not be difficult, since both systems are comparable in
size and shape, and both can be similarly recovered from feedstock by any
number of simple methods, including basic economical operations such as
filtration, centrifugation, decantation of solvent, and others.
In addition, the use of lyophilized CLECs permits routine handling and
storage of these materials prior to use (dry storage at room temperature
without refrigeration, for extended periods of time). Lyophilized CLECs
also allow for routine formulation by direct addition of solvents and
substrates of interest, without lengthy solvent exchange processes, or the
formation of amorphous suspensions. The lyophilized CLEC form extends the
general utility of the enzymes as catalysts to a broader spectrum of
enzymes and functional conditions.
A second advantage of a CLEC is that cross-linking of the crystallized
enzyme stabilizes and strengthens the crystal lattice and the constituent
enzyme molecules, both mechanically and chemically. As a result, a CLEC
may be the only means of achieving significant concentrations of active
enzyme catalyst in harsh aqueous, organic, near-anhydrous solvents, or in
aqueous-organic solvent mixtures. The use of enzymes as catalysts in
organic syntheses has been hampered by their tendency to denature in the
presence of non-aqueous solvents, and particularly, in mixtures of aqueous
and non-aqueous solvents (Klibanov, A. M., Trends in Biochemical Sciences,
14:141-144 (1989)). In CLECS, the restriction of conformational mobility
that leads to stability is provided by the inter-molecular contacts and
cross-links between the constituent enzyme molecules making up the crystal
lattice, rather than by the near-absence of water in the medium. As a
result, intermediate water concentrations can be tolerated by enzymes when
formulated as CLECs, as has previously not been possible (see Table 12).
In commercial applications, aqueous-organic solvent mixtures allow
manipulation of product formation by taking advantage of relative
solubilities of products and substrates. Even in aqueous media, enzyme
catalysts, immobilized or soluble, are subject to mechanical forces within
a chemical reactor that can lead to denaturation and a shortened
half-life. The chemical cross-links within the CLEC provide the necessary
mechanical strength (Quiocho and Richards, Proc. Natl. Acad. Sci. (USA)
52: 833-839 (1964)) that results in increased reactor life for the enzyme
catalyst.
A third advantage of a CLEC is that as a result of its crystalline nature,
a CLEC can achieve uniformity across the entire cross-linked crystal
volume. Crystalline enzymes as described herein are grown and cross-linked
in an aqueous environment and, therefore, the arrangement of molecules
within the crystal lattice remains uniform and regular. This uniformity is
maintained by the intermolecular contacts and chemical cross-links between
the enzyme molecules constituting the crystal lattice, even when exchanged
into other aqueous, organic or near-anhydrous media, or mixed
aqueous/organic solvents. In all of these solvents, the enzyme molecules
maintain a uniform distance from each other, forming well-defined stable
pores within the CLECs that facilitate access of substrate to the enzyme
catalysts, as well as removal of product. Uniformity of enzyme activity is
critical in industrial, medical and analytical applications where
reproducibility and consistency are paramount.
A fourth advantage of using a CLEC is that it should exhibit an increased
operational and storage half-life. Lattice interactions, even in the
absence of cross-linking, are known to stabilize proteins, due in part to
restrictions of the conformational degrees of freedom needed for protein
denaturation. In CLECS, the lattice interactions, when fixed by chemical
cross-links, are particularly important in preventing denaturation,
especially in mixtures of aqueous and non-aqueous solvents (Klibanov, A.
M., Trends in Biochemical Sciences 14: 141-144 (1989)). Enzymes that have
been in the crystalline state for months or years routinely retain a high
percentage of their catalytic activity. Cross-linked immobilized enzyme
crystals stored in anhydrous solvents will be even further protected from
microbial contamination and damage, which is a serious problem in storing
large quantities of protein in a nutrient rich, aqueous environment.
In the case of a lyophilized CLEC, the immobilized enzyme is stored in the
absence of solvent. That, and the stabilization achieved by cross-linking
allows for the storage in the absence of refrigeration for long periods of
time.
A fifth advantage of using a CLEC is that it should exhibit enhanced
temperature stability as a consequence of the cross-links stabilizing the
crystal lattice. Carrying out reactions at a higher temperature than that
used with conventional methods would increase reaction rates for the
chemical reactions of interest, both thermodynamically, and by enhancing
the diffusion rate into and out of the crystal lattice of CLECs. These
combined effects would represent a major improvement in reaction
efficiency, because they would maximize the productivity of a given
quantity of enzyme catalyst, which is generally the most expensive
component of the reaction process (Daniels, M. J., Methods in Enzymol.
136: 371-379 (1987)). The temperature stability exhibited by CLECs is
remarkable because most enzyme systems require mild reaction conditions.
CLECs would also be stabilized against denaturation by transient high
temperatures during storage.
A final advantage of use of a CLEC is that pores of regular size and shape
are created between individual enzyme molecules in the underlying crystal
lattice. This restricted solvent accessibility greatly enhances the metal
ion or cofactor retention characteristics of CLEC as compared to
conventionally immobilized enzymes and enzymes in solution. This property
of CLEC will permit the use of economically superior continuous-flow
processes in situations (see e.g. Oyama et. al. Methods in Enzymol. 136
503-516 (1987)) where enzyme would otherwise be inactivated by metal ion
or cofactor leaching. For example, in the thermolysin-mediated synthesis
of the dipeptidyl aspartame precursor, Z-L-Asp-L-Phe-OMe, conventionally
immobilized enzyme is known to lose catalytic activity in continuous-flow
column processes, in part through the leaching of calcium ions essential
for thermolysin activity. In practice, leaching of calcium ions has forced
the use of less efficient batch processes (Nakanishi et. al.,
Biotechnology 3: 459-464 (1985)). Leaching occurs when calcium ion
complexes are formed with substrate Z-L-Asp, in competition with the
natural calcium binding sites on the surface of the enzyme, resulting in
the loss of catalytic activity. The high density of enzyme, and the
correspondingly limited volume accessible to solvent in the interstices of
the CLECs, discourages the formation of the competing L-Asp-Ca.sup.++
complexes responsible for metal ion leaching.
In addition, crystallized, crosslinked antibodies, or CLACs, made by a
method similar to that used to produce CLECs, are the subject of the
present invention. As described with reference to CLECs, although
crosslinking contributes to the stability of a crystallized antibody, it
is neither necessary nor desirable in all instances. Crystallized
antibodies which are not subsequently crosslinked can be used in some
embodiments of the present invention. CLACs of the present invention have
the same advantages as described herein for CLECs. A particularly useful
advantage is the enhanced resistance to degradation (e.g., protease
degradation) of CLACs.
Preparation of CLECs--Enzyme Crystallization
In the method of the present invention, a cross-linked immobilized enzyme
crystal (or CLEC) is prepared as follows:
Enzyme crystals are grown by the controlled precipitation of protein out of
aqueous solution, or aqueous solution containing organic solvents.
Conditions to be controlled include, for example, the rate of evaporation
of solvent, the presence of appropriate co-solutes and buffers, and the pH
and temperature. A comprehensive review of the various factors affecting
the crystallization of proteins has been published by McPherson (Methods
Enzymol. 114: 112 (1985)). In addition, both McPherson and Gilliland (J.
Crystal Growth 90: 51-59 (1988)) have compiled comprehensive lists of all
proteins and nucleic acids that have been reported as crystallized, as
well as the conditions that lead to their crystallization. A compendium of
crystals and crystallization recipes, as well as a repository of
coordinates of solved protein and nucleic acid crystal structures, is
maintained by the Protein Data Bank (Bernstein et. al. J. Mol. Biol. 112:
535-542 (1977)) at the Brookhaven National Laboratory. Such references can
be used to determine the conditions necessary for the crystallization of a
given protein or enzyme previously crystallized, as a prelude to the
formation of an appropriate CLEC, and can guide the formulation of a
crystallization strategy for proteins that have not. Alternatively, an
intelligent trial and error search strategy (see eg., Carter, C. W. Jr.
and Carter, C. W., J. Biol. Chem. 254: 12219-12223 (1979)) can, in most
instances, produce suitable crystallization conditions for most proteins,
including, but not limited to, those discussed above, provided that an
acceptable level of purity can been achieved for these. The level of
purity required can vary widely from protein to protein. In the case of
lysozyme, for example, the enzyme has been crystallized directly from its
unpurified source, the hen egg-white (Gilliland, G. L., J. Crystal Growth
90: 51-59 (1988)). For use as CLECs in the method of this invention, the
large single crystals which are needed for X-ray diffraction analysis are
not required, and may, in fact, be undesirable because of diffusion
problems related to crystal size. Microcrystalline showers (ie., crystals
in the order of 10.sup.-1 mm in size/cross section) are suitable for CLECs
and are often observed, although seldom reported in the X-ray
crystallographic literature. Micro-crystals are very useful in the method
of this invention to minimize problems with diffusion (see eg., Quiocho,
F. A., and Richards, F. M., Biochemistry 5: 4062-4076 (1967)).
In general, crystals are produced by combining the protein to be
crystallized with an appropriate aqueous solvent or aqueous solvent
containing appropriate precipitating agents, such as salts or organics.
The solvent is combined with the protein at a temperature determined
experimentally to be appropriate for the induction of crystallization and
acceptable for the maintenance of protein stability and activity. The
solvent can optionally include co-solutes, such as divalent cations,
co-factors or chaotropes, as well as buffer species to control pH. The
need for co-solutes and their concentrations are determined experimentally
to facilitate crystallization. In an industrial scale process, the
controlled precipitation leading to crystallization can best be carried
out by the simple combination of protein, precipitant, co-solutes, and
optionally buffers in a batch process. Alternative laboratory
crystallization methods, such as dialysis, or vapor diffusion can also be
adapted. McPherson (Methods Enzymol. 114: 112 (1985)), and Gilliland (J.
Crystal Growth 90: 51-59 (1988)) include a comprehensive list of suitable
conditions in their reviews of the crystallization literature.
Occasionally, incompatibility between the cross-linking reagent and the
crystallization medium might require exchanging the crystals into a more
suitable solvent system.
Many of the proteins for which crystallization conditions have already been
described in the literature, have considerable potential as practical
enzyme catalysts in industrial and laboratory chemical processes, and are
directly subject to formulation as CLECs within the method of this
invention. Table 1 is a sampling of enzymes that have already been
crystallized. Note that the conditions reported in most of these
references have been optimized for the growth of large, diffraction
quality crystals, often at great effort. Some degree of adjustment of
conditions for the smaller crystals used in making CLECS might be
necessary in some cases.
TABLE 1
__________________________________________________________________________
Microbial or References
Enzyme biological source
(including those cited therein)
__________________________________________________________________________
alcohol dehydrogenase
horse liver Eklund et al., J. Mol. Biol.
146: 561-587 (1981)
alcohol oxidase
Pichia pastoris
Boys et al., J. Mol. Biol.
208: 211-212 (1989)
Tykarska et al., J. Protein Chem.
9: 83-86 (1990)
aldolase rabbit muscle Eagles et al., J. Mol. Biol.
(fructose-bisphosphate) 45: 533-544 (1969)
Heidmer et al., Science
171: 677-680 (1971)
calf muscle Goryunov et al., Biofizika
14: 1116-1117 (1969)
human muscle Millar et al., Trans. Roy. Soc. Lond.
B293: 209-214 (1981)
Drosophila melanogaster
Brenner et al., J. Biol. Chem.
257: 11747-11749 (1982)
aldolase (PKDG)
Pseudomonas putida
Vandlen et al., J. Biol. Chem.
248: 2251-2253 (1973)
alkaline phosphatase
Escherichia coli
Sowadski et al., J. Mol. Biol.
150: 245-272 (1981)
asparaginase
Erwinia caratova
North et al., Nature
224: 594-595 (1969)
Escherichia coli
Epp et al., Eur. J. Biochem.
20: 432-437 (1971)
Escherichia coli
Yonei et al., J. Mol. Biol.
110: 179-186 (1977)
Proteus vulgaris
Tetsuya et al., J. Biol. Chem.
248: 7620-7621 (1972)
carbonic anhydrase
human erythrocyte (C)
Kannan et al., J. Mol. Biol.
12: 740-760 (1965)
human erythrocyte (B)
Kannan et al., J. Mol. Biol.
63: 601-604 (1972)
bovine erythrocyte
Carlsson et al., J. Mol. Biol.
80: 373-375 (1973)
catalase horse erythrocyte
Glauser et al., Acta Cryst.
21: 175-177 (1966)
Micrococcus luteus
Marie et al., J. Mol. Biol.
129: 675-676 (1979)
Penicillium vitale
Vainshtein et al., Acta Cryst.
A37: C29 (1981)
bovine liver Eventoff et al., J. Mol. Biol.
103: 799-801 (1976)
creatine kinase
bovine heart Gilliland et al., J. Mol. Biol.
170: 791-793 (1983)
rabbit muscle McPherson, J. Mol. Biol.
81: 83-86 (1973)
glutaminase
Actenobacter glutanimasificans
Wlodawer et al., J. Mol. Biol.
99: 295-299 (1975)
Pseudomonas 7A Wlodawer et al., J. Mol. Biol.
112: 515-519 (1977)
glucose oxidase
Aspergillus niger
Kalisz et al., J. Mol. Biol.
213: 207-209 (1990)
.beta.-lactamases
Staphylococcus aureus
Moult et al., Biochem J.
225: 167-176 (1985)
Bacillus cereus
Sutton et al., Biochem J.
248: 181-188 (1987)
lacate dehydrogenase
porcine Hackert et al., J. Mol. Biol.
78: 665-673 (1973)
chicken Pickles et al., J. Mol. Biol.
9: 598-600 (1964)
dogfish Adams et al., J. Mol. Biol.
41: 159-188 (1969)
Bacillus stearothermophilus
Schar et al., J. Mol. Biol.
154: 349-353 (1982)
lipase Geotrichum candidum
Hata et al., J. Biochem.
86: 1821-1827 (1979)
horse pancreatic
Lombardo et al., J. Mol. Biol.
205: 259-261 (1989)
Mucor meihei Brady et al., Nature
343: 767-770 (1990)
human pancreatic
Winkler et al., Nature
343: 771-774 (1990)
luciferase Firefly Green, A. A., et al., Biochem. Biophys. Acta.
20: 170 (1956)
luciferase Vibrio harveyii
Swanson et al., J. Biol. Chem.
260: 1287-1289 (1985)
nitrile hydratase
Brevibacterium R312
Nagasawa et al., Biochem. Biophys. Res.
Commun.
139: 1305-1312 (1986)
P. chlororaphis B23
Nagasawa et al., Eur. J. Biochem.
162: 691-698 (1987)
peroxidase horseradish Braithwaite et al., J. Mol. Biol.
106: 229-230 (1976)
horseradish roots (Type E4)
Aibara et al., J. Biochem.
90: 489-496 (1981)
Japanese radish
Morita, Acta Cryst.
A28: S52 (1979)
peroxidase (chloride)
Caldaromyces fumago
Rubin et al., J. Biol Chem.
257: 7768-7769 (1982)
peroxidase (cytochrome)
Sarchomyces cerevisae
Poulos et al., J. Biol. Chem.
253: 3370-3735 (1978)
peroxidase (glutathione)
bovine erythrocyte
Ladenstein et al., J. Mol. Biol.
104: 877-882 (1979)
subtilisin Bacillus subtilis (Novo)
Drenth et al., J. Mol. Biol.
28: 543-544 (1967)
Bacillus amyloliquefaciens (BPN')
Wright et al., Nature
221: 235-242 (1969)
Bacillus subtilis (Carlsberg)
Petsko et al., J. Mol. Biol.
106: 453-456 (1976)
superoxide dismutase
bovine Richardson et al., J. Biol Chem.
247: 6368-6369 (1972)
spinach Morita et al., J. Mol. Biol.
86: 685-686 (1974)
Saccharomyces cerevisiae,
Beem et al., J. Mol. Biol.
Escherichia coli
105: 327-332 (1976)
Bacillus stearothermophillus
Bridgen et al., J. Mol. Biol.
105: 333-335 (1976)
Pseudomonas ovalis
Yamakura et al., J. Biol Chem.
251: 4792-4793 (1976)
thermolysin
Bacillus thermoproteolyticus
Matthews et al., Nature New Biol.
238: 37-41 (1972)
urease jack bean Sumner, J. B., J. Biol. Chem.
69: 435 (1926)
xylose isomerase
Streptomyces rubiginosus
Carrell et al., J. Biol. Chem.
259: 3230-3236 (1984)
Arthrobacter B3728
Akins et al., Biochym. Biophys Acta
874: 375-377 (1986)
Streptomyces olivochromogenes
Farber et al., Protein Engineering
1: 459-466 (1987)
Streptomyces violaceoniger
Glasfeld et al., J. Biol. Chem.
263: 14612-14613 (1988)
Actinoplanes missouriensis
Rey et al., Proteins: Struc. Func. Genet.
4: 165-172 (1988)
__________________________________________________________________________
Preparation of CLECs--Cross-linking Reaction
Once crystals are grown in a suitable medium, they can be cross-linked.
Cross-linking results in stabilization of the crystal lattice by
introducing covalent links between the constituent enzyme molecules in the
crystal. This makes possible the transfer of enzyme into an alternate
reaction environment that might otherwise be incompatible with the
existence of the crystal lattice, or even with the existence of intact
undenatured protein. Cross-linking can be achieved by a wide variety of
bifunctional reagents, although in practice, simple, inexpensive
glutaraldehyde has become the reagent of choice. (For a representative
listing of other available cross-linking reagents, one can consult, for
example, the 1990 catalog of the Pierce Chemical Company). Cross-linking
with glutaraldehyde forms strong covalent bonds between primarily lysine
amino acid residues within and between the enzyme molecules in the crystal
lattice that constitute the crystal. The cross-linking interactions
prevent the constituent enzyme molecules in the crystal from going back
into solution, effectively insolubilizing or immobilizing the enzyme
molecules into microcrystalline (ideally 10.sup.-1 mm) particles. The
macroscopic, immobilized, insolubilized crystals can then be readily
separated from the feedstock containing product and unreacted substrate by
simple procedures such as filtration, decantation, and others. They can
also be used in CLEC packed columns in continuous flow processes, where
they exhibit enhanced cofactor and metal ion retention properties.
By the method of this invention, CLECs are obtained for use as enzyme
catalysts in existing and novel environments. The enhanced stability of
the CLECs, which results from the cross-linking reaction, makes it
possible to transfer the CLEC into a solvent (e.g., aqueous, organic or
near-anhydrous solvents, or a mixture of these), in which it would
otherwise be incompatible, and to carry out chemical reactor operation at
elevated temperatures of extremes of pH. The macroscopic CLEC catalyst
particles can also be readily manipulated, allowing recovery from
feedstock by simple methods, such as filtration, centrifugation, or
decantation of solvent. In addition, these can be used in packed columns
in continuous flow processes.
Preparation of CLECs--Lyophilization
A suspension of one volume of cross-linked thermolysin crystals in ten
volumes of demineralized water at pH 7.0 was lyophilized overnight using a
VirTis Model #24 lyophilizer. Lyophilized crystals were stored at room
temperature or at 4.degree. C. prior to reconstitution, which was
accomplished by adding ten volumes of the solvent of choice directly onto
crystals taken from storage. Re-hydrated crystals were reconstituted in 10
mM calcium acetate buffer at pH 7.0 for the FAGLA cleavage experiments.
Reconstituted lyophilized CLECs were routinely stored at room temperature.
In contrast, soluble enzyme required storage at -70.degree. C. to maintain
specific activity longer than a week. This protocol was used for all the
enzymes described in the exemplification included here.
Synthesis of Aspartame Precursor with Thermolysin CLEC
The method of the present invention, by which cross-linked crystal enzymes
are produced, is described below and exemplified by the production of
cross-linked immobilized enzyme crystals of thermolysin for use in the
production of the dipeptidyl precursor of aspartame, in ethyl acetate,
which is a near-anhydrous organic, solvent. Thermolysin, a protein which
has been crystallized and whose structure has been solved at 1.6 .ANG.
resolution (Holmes and Matthews, J. Mol. Biol. 160: 623-639 (1982)), is
one example of an enzyme which can be used as a CLEC in the present
method. Thermolysin is used in the manufacture of the artificial sweetener
aspartame (Isowa et. al. U.S. Pat. No. 4,436,925 (1984); Lindeberg, J.
Chem. Ed. 64: 1062-1064 (1987); Nakanishi et. al., Biotechnology 3:
459-464 (1985); Oyama, et. al., Methods in Enzymol. 136: 503-516 (1987)).
At the present time, most aspartame appears to be produced by a
conventional synthetic chemistry approach, although use of conventionally
immobilized thermolysin in near-anhydrous media has produced encouraging
results (Oyama et. al., J. Org. Chem. 46: 5242-5244 (1981); Nakanishi et.
al., Biotechnology 3: 459-464 (1985)). Improvement in the enzymatic
approach to aspartame production, such as is possible through use of the
present method, would make it competitive with the presently-used method,
both in terms of convenience and cost (Oyama, et. al., Methods in Enzymol.
136: 503-516 (1987)).
Assessment of Thermolysin CLECs
The method of the present invention has also been used to produce
thermolysin CLECs which have been assessed as to their pH dependence and
stability, stability at elevated temperature, resistance to exogenous
proteolysis and stability in the presence of an organic solvent.
Thermolysin CLECs were compared to soluble thermolysin, as described in
detail in Example 2 and FIGS. 1-4. Results of the assessment showed the
following:
1. As to pH dependence and stability, both forms demonstrate maximum
activity at pH 7 and demonstrate similar activity in the acidic range. In
the alkaline pH range, the CLEC maintains maximum activity to pH 10; the
soluble thermolysin has 75% activity at pH 8.5, only 25% activity at pH 9
and is completely inactive at pH 9.5.
2. The additional stabilization achieved in CLECs results in enzymatic
activity at higher temperatures than is possible with soluble thermolysin.
Enhanced stability of CLEC thermolysin at lower temperatures makes storage
simpler than it is for the soluble enzyme. Thermal stability and
resistance to autolysis was also demonstrated for thermolysin CLECs, which
retained maximum activity after five days of incubation at 65.degree. C.
In contrast, soluble thermolysin lost 50% of its initial activity after
two hours incubation and demonstrated negligible activity after 24 hours
incubation at 65.degree. C.
3. Enzymatic activity of thermolysin CLECs was unaffected by four days'
incubation in the presence of the powerful streptococcal protease,
Pronase.RTM.. In contrast, soluble thermolysin was rapidly degraded and
lost all activity after 90 minutes incubation.
4. Thermolysin CLECs and soluble thermolysin exhibited markedly different
stability in the presence of organic solvents, as shown in Table 12. The
thermolysin CLECs retained greater than 95% maximum activity following
incubation with all organic solvents assessed. Additional work with
soluble and CLEC thermolysin catalysed synthesis is described in Example 6
and FIG. 5.
These features of thermolysin CLECs and other enzyme CLECs make them
particularly useful, since they are easier to store, more stable and less
easily inactivated or degraded than corresponding soluble enzymes.
Assessment of Elastase CLECs
The method of the present invention has also been used to produce elastase
CLECs which have been assessed as to their activity and resistance to
exogenous proteolysis. Elastase CLECs were compared to soluble elastase,
as described in detail in Example 4 and FIGS. 6 and 7. Results of the
assessment demonstrated the following:
1. Elastase CLECs retain approximately 50% activity compared to soluble
enzyme.
2. Soluble elastase was rapidly degraded by protease. Activity of soluble
elastase was reduced to 50% of initial activity following ten minutes
incubation in the presence of protease. After one hour incubation the
soluble enzyme had lost more than 90% of its initial activity. In contrast
the enzymatic activity of the elastase CLEC was unaffected by incubation
with protease.
Assessment of Esterase CLECs
The method of the present invention has also been used to produce esterase
CLECs which have been assessed as to their activity and resistance to
exogenous proteolysis. Esterase CLECs were compared to soluble esterase,
as described in detail in Example 5 and FIGS. 8 and 9. Results of the
assessment demonstrated the following:
1. Esterase CLECs retain approximately 50% activity compared to soluble
enzyme.
2. Soluble esterase was highly susceptible to proteolytic degradation.
Activity of soluble esterase was reduced to 50% of initial activity
following ten minutes incubation in the presence of protease. After one
hour incubation the soluble enzyme had lost more than 90% of its initial
activity. In contrast the enzymatic activity of the esterase CLEC was
unaffected by incubation with protease.
Assessment of Lipase CLECs
The method of the present invention has also been used to produce lipase
CLECs which have been assessed as to their activity. Lipase CLECs were
compared to soluble lipase, as described in detail in Examples 6 and 10
and FIG. 9. Results of the assessment demonstrated that G. candidum and C.
cylindracea lipase CLECs retain significant activity, compared to soluble
enzyme (Examples 6 and 10) and that the porcine pancreatic lipase retained
activity to a limited extent. (Example 11)
Assessment of Lysozyme CLECs
The method of the present invention has also been used to produce lysozyme
CLECs which have been assessed as to their activity and resistance to
exogenous proteolysis. Lysozyme CLECs were compared to soluble lysozyme as
described in detail in Example 7 and FIG. 11. Results of the assessment
demonstrated that lysozyme CLECs retain approximately 50% activity
compared to soluble enzyme.
Assessment of Asparaginase CLECs
The method of the present invention has also been used to produce
asparaginase CLECs which have been assessed as to their activity.
Asparaginase CLECs were compared to soluble asparaginase, as described in
detail in Example 8 and FIG. 12. Results of the assessment demonstrated
the following asparaginase CLECs retain approximately 77% activity
compared to soluble enzyme.
Assessment of Urease CLECs
The method of the present invention has also been used to produce urease
CLECs which have been assessed as to their activity. Urease CLECs were
compared to soluble urease, as described in detail in Example 9 and FIGS.
13-16 and, in addition, urease CLEC activity in an aqueous buffer was
compared with its activity in sera, which is a biologically relevant
medium. (Example 9 and FIG. 17) Results of the assessment demonstrated
that urease CLECs retain significant enzymatic activity and that there was
comparable urease activity in aqueous buffer and in sera.
General Applicability of CLECs
As disclosed here, CLECs represent a novel technology with broad use in
many areas, including, but not limited to, industrial scale syntheses,
laboratory tools, biosensors, and medical applications. Examples of
various systems using conventionally immobilized enzyme methods in their
execution are given in Tables 2-5 below. One skilled in the art should be
able to adapt these, and similar systems, to the CLEC technology disclosed
in this application. To illustrate this, specific examples are discussed
in more detail from each of the categories listed.
Table 2 below lists examples which use conventionally immobilized enzymes
in an industrial process, which examples can be readily adapted to the
CLEC technology disclosed here.
TABLE 2
__________________________________________________________________________
Production or References
Enzyme application
Substrates (including those cited)
__________________________________________________________________________
thermolysin aspartame precursor
Z-Asp, L-Phe-OMe
Oyama et al., J. Org. Chem.
46: 5242-5244 (1981)
Nakanishi et al., Trends in
Biotechnology 3: 459-464 (1985)
subtilisin aspartame L-Asp-L-Phe, OMe
Davino, A. A., U.S. Pat. No.
4293648 (1981)
lipase cocoa fat palm oils Harwood. J., Trends in
substitutes Biochemical Sciences
14: 125-126 (1989)
Macrae, A. R., J. Am. Oil Chem Soc.
60: 291-294 (1983)
nitrile hydratase,
acrylamide
acrylonitrile
Nagasawa, T. and Yamada, H.,
nitrilases, amidase Trends in Biotechnology
7: 153-158 (1989)
amino acylase (fungal)
amino acid
N-acyl-D,L amino acids
Schmidt-Kastner, G. & Egerer,
amino acid esterase,
resolution
ester of D,L amino
P. in Biotechnology vol 6a:
subtilisin acids 387-421 (1984) and references
amidases amides of D,L amino
therein.
hydantionases acids
specific dehydrogenases
hydantoins Fusee, M. C., Methods in
amino peptidase a-hydroxycarboxylic
Enzymology 136: 463 (1987)
transaminase acids
Fusee, M. C., Methods in
Enzymology 136: 463 (1987)
amino acid amino acid
keto or hydroxy acids
dehydrogenase + formate
production: general Rozzell, J. D., Methods in
dehydrogenase Enzymology 136: 479 (1987)
amino acid Enzymes in Industry; Ed
production specific Gerhartz. W., VCH Press 1990
L-aspartase L aspartic acid
fumarate/fumaric acid
L-aspartate 4-
L-alanine L-aspartic acid,
decarboxylase ammonium fumarate
aspartase + L aspartate 4
decar-boxylase
L-lysine D,L-a amino e-
ACL hydrolase caprolactam (ACL)
L-ACT hydrolase
L-cysteine
DL-2amino2 thiazoline
4carboxylic acid
L-isoleucine
L-methionine
lyase L-phenylalanine
cinnamate
L-tryptophan synthetase
L-tryptophan
indole, L-serine
L-valine
fumarase L-malic acid
fumarate
hydantoinase
D n carbamoyl p-
5p-hydroxy hydantoin
hydroxy-phenyl
glycine
lipases, esterases
resolution of
synthetic chemistry
Jones, J. B., Tetrahedron
racemates by 42: 3351-3403 (1988)
stereoselective Butt, S. and Roberts, S. M.,
synthesis Natural Product Reports
489-503 (1986), and references cited
therein for a more comprehensive
review of this area
fumarase L-maltic acid
fumaric acid
Chibata et al., Methods in
Enzymology 136: 455 (1987)
lactase, .beta.-galactosidase
disaccharide
lactose & N-acetyl
Larsson et al., Methods in
synthesis eg
galactosamine
Enzymology 136: 230 (1987)
galactosyl-N-acetyl
galactosamine
lipase, esterase
L-menthol 4 isomer mix
Fukui, S., Tanaka, A., Methods
in Enzymology 136: 293 (1987)
amidases D-valine D,L amino acid amide
Schmidt-Kastner, G. & Egerer,
(intermediate for P. in Biotechnology vol 6a:
pyrethroid 387-421 (1984) and references
insecticide fluvinate)
therein.
lipase (Candida
R(+)2 phenoxy-
2 chloro propionic acids
Biocatalysts in Organic
cylindricea)
propionic acids Syntheses eds Tramper, van de
(herbicides) Plas & Linko; Proceedings of
International Symposium in
Netherlands 1985
lipases, esterases,
organic synthesis Jones, J. B. Tetrahedron
amidases, aldolases
monoglycerides 42: 3351-3403 (1988)
peptides Butt, S. and Roberts, S. M.,
protease, peptidases Natural Product Reports
489-503 (1986), and references cited
yeast lipase
2(p-chlorophenoxy)
resolution of racemic
therein for a more comprehensive
propionic acid:
ester review of this area.
herbicide
strictodine synthetase
alkaloid production Pfitzner et al., Methods in
eg strictosidine Enzymology 136: 342 (1987)
penicillin acylase
6-amino penicillin G or V
Enz Eng 6: 291 (1982)
penicillin amidase
penicillinanic acid Enz Eng 8: 155
and 7-ADCA
hydroxysteroid
steroid Carrea et al., Methods in
dehydrogenases
transformations Enzymology 136: 150 (1987)
5'phosphodiesterase,
5'-ribonucleotides Keller et al., Methods in
nucleases Enzymology 136: 517 (1987)
esterase .beta.-lactam precursor
corresponding diesters
Japanese patent application:
(chiral mono esters 82-159, 493 (1981) Biseibutsu
eg .beta.amino glutaric
Company
acid monoalkyl ester)
lipases .beta.-blockers Kloosterman, M et al., Trends in
Biotechnology 6: 251-256
__________________________________________________________________________
(1988)
Production of Acrylamide Using CLEC Technology
The following is a description of one use of the method of the present
invention: the adaptation of acrylamide production from immobilized cells
which overproduce nitrile hydratase enzyme (Nagasawa, T. and Yamada, H.,
Trends in Biotechnology 7: 153-158 (1989)) to the CLEC technology
previously disclosed herein.
Industrial scale production of acrylamide, an important commodity chemical,
has been described by Yamada and collaborators (Nagasawa, T. and Yamada,
H., Trends in Biotechnology 7: 153-158 (1989)). Kilotons of acrylamide per
year are produced in chemical reactors loaded with entrapped cells
selected as overproducers of the enzyme nitrile hydratase. Nitrile
hydratase has also been reported as purified and crystallized from two
sources, Brevibacterium R312 (Nagasawa et. al., Biochem. Biophys. Res.
Commun. 139: 1305-1312 (1986) and P. chlororaphis B23 (Nagasawa et. al.,
Eur. J. Biochem. 162: 691-698 (1987). As disclosed here, these crystalline
enzymes can each be immobilized by crosslinking with glutaraldehyde or
other suitable crosslinking reagent to produce a CLEC. The nitrile
hydratase CLECs can then be used in a conventional reactor, in place of
the entrapped cells currently used. Adapting this process to CLEC
technology leads to immediate advantages. These include; reduced plant
size and improved throughput resulting from the enhanced activity per unit
volume implicit in the higher enzyme concentration in the CLECs, and
improved substrate and product diffusion rates; reduction in undesired
contamination and side reactions, resulting from the higher purity of
CLECS; and reduced sensitivity to microbial contamination in the absence
of cells. In addition, there are other benefits available only to a
CLEC-based method. These benefits include: higher temperature operation to
improve reaction rates; the ability to operate in aqueous, organic and
near-anhydrous solvents, allowing optimization of the acrylamide
production reaction; and enhanced half-life in operation and storage,
resulting from the higher chemical and mechanical stability of CLECs,
particularly in unconventional solvents.
Medical Applications of CLEC Technology
The method of the present invention and an appropriately selected CLEC or
set of CLECs can also be used for medical applications. A CLEC or a set of
CLECs can be used, for example, to remove a component of a fluid, such as
blood, generally by altering the component, and thus, converting it to a
substance not detrimental to an individual or which can be removed by
normal body processes (e.g., via detoxification, or degradation in the
liver, excretion via the kidneys). In this application, an
appropriately-selected CLEC or set of CLECs is brought into contact with
body fluid, which contains the component to be altered, or a reactant
(product or substrate) of a reaction in which the component participates,
upon which the enzyme in the CLEC acts. As a result, the enzyme is able to
act upon the component to be altered or with another substance which is a
product of a reaction in which the component to be altered participates.
The activity of the enzyme results in direct alteration of the component
to be removed or in alteration of the product of the reaction in which the
component participates (thus making continuation of the reaction
impossible). This can be carried out through the use of an extracorporeal
device which includes an appropriately-selected CLEC or set of CLECs and a
retaining means which is made of a material; such as a porous material on
which a CLEC is retained or a tube in which a CLEC is present, which
allows contact between the component itself or the substance in the fluid
which is a product of a reaction in which the component to be altered
participates.
This might also be achieved by the insertion of an appropriate CLEC into a
suitable body compartment, such as the peritoneum or a lymph node, where
the CLEC would have access to bodily fluids. This insertion might be done
surgically, or by injection of the CLEC slurry. Direct injection of CLEC
into the blood stream would not be appropriate, given the high risk of
embolism.
The use of appropriate CLECs in this area might serve as an alternative to
genetic methods in enzyme replacement therapy to correct a natural
deficiency, such as, e.g. phenylketonuria.
Table 3 illustrates some of the medical applications in which CLECs could
be used. For the majority of these cases, the extra-corporeal treatment is
still in the research phase, but the benefits that CLECs offer could
provide novel treatments in areas in which there was previously no
alternative treatment.
TABLE 3
__________________________________________________________________________
Enzyme
employed Removal of:-
Disease/patients treated
References
__________________________________________________________________________
asparaginase
asparagine
leukemia Klein, M., Langer, R., Trends in
(Removal of asparagine, an
Biotechnology 4: 179-185
important cancer nutrient, harms
(1986) and references therein
leukemia cells ehich cannot
manufacture the essential amino
Chang, T. M. S., Methods in
acid - asparagine; normal cells
Enzymology 137: 444-457
can manufacture asparagine and so
(1987) & references therein
are unaffected by this treatment.)
heparinase
heparin deheparinization for
Langer, R., et al., Science
hemoperfusion patients eg kidney
217: 261-263 (1982)
dialysis
bilirubin oxidase
bilirubin
neo-natal jaundice
Lavin, A., et al., Science
230: 543-545 (1985)
carboxypeptidase
methotrexate
chemotherapy patients
Pitt, A. M., et al., Appl.
Biochem. Biotechnol. 8: 55-68 (1983)
tyrosinase
aromatic amino
liver failure exhibiting
Chang, T. M. S., Sem. Liver
acids pathological elevations of amino
Dis. Ser. 6: 148 (1986)
acids
phenylalanine
phenylalanine
phenylketonuria and liver failure
Ambrus, C. M., et al.,
ammonium lyase J. Pharm. & Exp. Ther.
224: 598-602 (1983)
multi-enzyme
urea (converted
detoxification for chronic renal
Chang, T. M. S., Methods in
system including:
into glutamic and
failure patients
Enzymology 137: 444-457
urease, glutamate
other amino acids, (1987) & references therein
dehydro-genase,
via ammonia)
glucose, Chang, T. M. S., Enzyme Eng
dehydrogenase & a 5: 225 (1980)
transaminase
arginase arginine familial hyperargininaemia
Kanalas, J. J. et al., Biochem.
Med. 27: 46-55 (1982)
glutamate
ammonia liver failure Maugh, T. H., Science
dehydrogenase & 223: 474-476 (1984)
ammonia
__________________________________________________________________________
A particular application of the present method is the heparin lyase system
for blood deheparinization (Bernstein et al., Methods in Enzymology 137:
515-529 (1987)), which is discussed below.
All extracorporeal devices perfused with blood, such as kidney dialysis,
continuous arteriovenous hemofiltration or extracorporeal membrane
oxygenators, require heparinization of the patient to avoid blood
clotting. However, heparinization of the patient leads to hemorrhagic
complications and remains a threat to human safety. These problems
increase as the perfusion times increase, for example with the membrane
oxygenator, and can lead to serious bleeding. After extracorporeal
therapy, heparin may be removed from blood by employing a heparinase
device at the effluent of the extracorporeal device which eliminates all
heparin from the blood returned to the patient and thus avoids the current
problems of heparinization.
Published research (Langer et al. Science 217: 261-263 (1982)); Bernstein
et al., Methods in Enzymology 137: 515-529 (1987)), details the problems
presented by conventionally immobilized enzymes used in extracorporeal
devices. The principal problem is that conventional immobilization results
in a low retention of enzyme activity per unit volume, thus requiring a
large volume of immobilized enzyme to perform the necessary
heparinization. This volume is too large to be of practical use in humans.
However, the high retention of activity per unit volume in CLECs, due to
the lack of inert support circumvents this problem and offers a practical
solution to deheparinization of humans. The enhanced stability of CLECs
will reduce the disassociation of enzyme from the crosslinked crystal.
This is superior to the less stable, conventionally immobilized enzymes,
because immune responses resulting from enzyme leakage will be reduced.
CLECs temperature stability prevents denaturation of the enzyme due to
high transient temperatures during storage; it is likely that CLEC may
retain high activity, even when stored at room temperature. In addition,
CLECs will be cheaper and more convenient to use than their conventionally
immobilized counterparts because of their longer operational and storage
lifetimes.
CLECs made by the present method can be used for additional medical
purposes, both therapeutic and diagnostic. As described herein, enzymes
which have potential for such uses have been crystallized and crosslinked
and assessed as to their enzymatic activity and stability under various
conditions. For example, lipase CLECs have been produced and shown to
retain significant enzymatic activity. Such lipase CLECs can be used, for
example, to treat individuals with pancreatic insufficiency and/or fat
malabsorption conditions, in which lipase secretion is abnormally low.
This can be associated with steatorrhea, essential fatty acid deficiency,
loss of a high calorie source (fat) or a fat-soluble vitamin deficiency.
Presently available approaches to lipase supplementation have numerous
shortcomings, which limit their effectiveness. For example, gastric acid
inactivation of enzyme supplements or digestion by proteases of
lipase-supplementation agents may occur, reducing the available amount of
the agent(s) used. Possible supplementation strategies include high doses
of pancreatic enzymes, use of pH sensitive enteric-coated microspheres and
capsules, gastric acid modulation and use of acid resistant lipases (which
presently are unavailable). Lipase CLECs can be used as therapeutic agents
or drugs and, in this context, have several key advantages: they are
stable to exogenous proteolysis, stable to pH levels which would
inactivate or destroy other enzyme forms, stable to heat and solvents and
easily stored because they can be lyophilized; as described herein. Other
therapeutic uses include administration of or treatment with urease. CLECs
and xanthine oxidase CLECs (e.g., in treating hyperuriocosuria, resulting
in conversion, in the GI tract, of purines to uric acid, followed by
excretion of the uric acid.
In therapeutic applications, design and use of CLECs which release the
enzyme over time (e.g., slow or controlled release) might prove
advantageous, such as to provide the activity over time or to delay its
release (e.g., to allow the enzyme to pass through harsh pH conditions in
the stomach by being protected in CLEC form and then being released).
Production of CLECs in which crosslinking and/or crystallization is
designed to permit slow or controlled release would provide useful agents.
Additional Applications of CLEC Technology: Biosensors
A CLEC or a set of CLECs can be used as a component of a sensor, referred
to as a biosensor, useful for detecting and/or quantitating an analyte of
interest in a fluid, such as body fluid (e.g., blood, urine), chemical and
laboratory reaction media, organic media, water, culture medium and
beverages. In some instances, the fluid in question can be a gas, as in an
alcohol breath analyzer (Barzana, E., Klibanov, A., and Karell, M., NASA
Tech Briefs 13:104 (1989)). In this application an appropriately-selected
CLEC or set of CLECs is brought into contact with a fluid to be analyzed
for the analyte of interest. The analyte of interest can be measured
directly (e.g., blood glucose level) or indirectly (e.g., by detecting or
quantitating a substance which is a reactant (product or substrate) in a
reaction in which the analyte of interest participates). In either case,
the CLEC is able to act upon the analyte or the substance which is a
reactant in a reaction in which the analyte also participates. The
activity of the enzyme results in a detectable change (e.g., change in pH,
production of light, heat, change in electrical potential) which is
detected and/or quantitated by an appropriate detecting means (e.g., pH
electrode, light or heat sensing device, means for measuring electrical
charge) (Janata, J., et al., Anal. Chem. 62: 33R-44R (1990)). Any means
useful for detecting the change resulting from the enzyme-catalyzed method
can be used. A biosensor of the present invention includes a CLEC or set
of CLECs and a retaining means for the CLEC which allows contact between
the CLEC(s) and the analyte of interest or the substance in the fluid
which is a reactant in the reaction in which the analyte of interest
participates.
Table 4 illustrates some of the biosensor applications in which CLECs could
be used. Currently immobilized enzymes are used in these applications, but
suffer from low stability, low enzyme density, short lifetimes and lack of
reproducibility. These examples can be readily adapted to the CLEC
technology disclosed here.
TABLE 4
__________________________________________________________________________
Enzyme Employed
Detection of:-
Application
Reference
__________________________________________________________________________
glucose oxidase
glucose diabetics Daniles, B., Mossbach, K.,
Methods in Enzymology
137: 4-7 (1987)
Hall, E "Biosensors" Open
University Press (1990)
Taylor, R., Proceed.
Biotechnology Conference
1989; 275-287
Anthony et al.,
"Biosensors, Fundamentals
and Applications", Oxford
University Press (1987)
creatinine deiminase
creatinine kidney function
Tabata, M. et al., Anal.
Biochem. 134: 44 (1983)
urease urea kidney function
Hsuie, G. H. et al.,
Polym. Mater. Sci. Eng.
57: 825-829 (1987)
Kobos, et al. Anal. Chem.
60: 1996-1998 (1988)
lactate oxidase &
lactate clinical applications
Blaedel, W. J. & Jenkins, R.
dehydrogenase A., Anal. Chem. 48(8): 1240 (1976)
Sagaguchi, Y., et al., J.
Appl. Biochem 3: 32 (1981)
glucose-6-pyruvate
Glucose-6-phosphate,
diabetics and other
ibid as glucose oxidase
dehydrogenase
suctrose and ATP
medical
alcohol dehydrogenase,
ethanol & other
breathalysers and
Romette, J. L. et al.,
alcohol oxidase
alcohols; acetic, formic
industrial applications
Methods in Enzymology
acids 137: 217-225 (1987)
Ho, M. H., Methods in
Enzymology 137: 271-288 (1987)
.beta.-fructosidase
sucrose industrial applications
Romette, J. L. et al.,
Methods in Enzymology
137: 217-225 (1987)
cholesterol oxidase
cholesterol
cholesterol testing
Satoh, I., Methods in
Enzymology 137: 217-225 (1987)
catalase uric acid, cholesterol
atherosclerotic and
Satoh, I., Methods in
other medical
Enzymology 137: 217-225 (1987)
carboxy peptidase
methotrexate
cancer ibid as glucode oxidase
carbonic anhydrase
carbon dioxide
industrial, laboratory &
ibid as glucose oxidase
environmental
applications
L-amino acid oxidase
amino acids
medical and industrial
ibid as glucose oxidase
.beta.-lactamase
penicillin medical Anzai et al., Bull. Chem.
penicillinase Soc. Jpn. 60: 4133-4137 (1988)
alkaline phosphatase
phosphate metabolite monitoring
ibid as glucose oxidase
nitrate/nitrite reductase
nitrates & nitrites
metabolite and food
ibid as glucose oxidase
monitoring
arylsulfatase
sulfate metabolite monitoring
ibid as glucose oxidase
succinate succinate industrial ibid as glucose oxidase
dehydrogenase
bacterial luciferase
FMNH.sub.2 and coupled
detection of 10.sup.-18
Wannlund J., et al.,
reactions molar quantities of
"Luminescent assays:
FMNH.sub.2 through
Perspectives in
measurement of photon
endocrinology and clinical
release chemistry"; Eds Serio, M.
and Pazzagli, M. 1: 125 (1982)
Kurkijarvi et al., Methods
in Enzymology 137: 171-181 (1987)
firefly luciferase
ATP and coupled
detection of 10.sup.-12
Kurkijarvi et al., Methods
reactions molar quantities of ATP
in Enzymology 137: 171-181 (1987)
through measurement of
Murachi et al., Methods in
photon release
Enzymology 137: 260-271
__________________________________________________________________________
(1988)
In the method of the present invention as it is carried out for the
analysis of samples in a biosensor, it is particularly desirable to
produce the largest possible detectable signal from the smallest possible
quantity of substrate and catalyst. In this regard, the CLEC technology
disclosed here is particularly attractive, since it achieves the highest
possible concentration of enzyme catalyst in a given volume.
Often, considerable efforts are made to couple an ultimate enzymatic
reaction of interest, either directly, or through suitable intermediates,
to the production of light by enzymes like luciferase (Kurkijarvi et al.,
Methods in Enzymol. 137: 171-181 (1988)). This is done in order to take
advantage of the unparalleled sensitivity and efficiency of photon
detection equipment, which allows for the detection of femtomolar
concentrations of enzyme reaction products under appropriate conditions.
Following this principle, biosensor systems have been designed, using
conventionally immobilized enzymes, to detect various substrates of
clinical and other interest. Light producing reactions have been coupled
to assay reactions detecting substrates like, D-glucose, L-lactate,
L-glutamate and ethanol, among others, at extremely low concentration.
With regard to this application, the luciferase enzyme from Vibrio harveyii
has been reported as crystallized (Swanson et al., J. Biol. Chem. 260:
1287-1289 (1985)). Crystals of this luciferase can be crosslinked with
glutaraldehyde or other suitable reagent to form a CLEC of luciferase. For
biosensor and analytical uses, a CLEC of luciferase offers many advantages
over conventionally immobilized enzyme. In a CLEC, the entire volume of
the luciferase CLEC would consist of light emitting enzyme. In a
conventionally immobilized enzyme system, however, as much as 95% of the
total volume is taken up by "inert" carrier material, which more likely
functions as an absorber of the light emitted by enzyme. In addition, the
enhanced stability of CLECs should facilitate storage at room temperature,
and also makes possible novel sensing applications in harsh environments
and elevated temperatures.
CLACs of the present invention should be useful for diagnostic and
therapeutic purposes (e.g., delivery of an agent (such as a label or a
cytotoxic agent) to a defined cell type (one recognized by the antibody).
Additional Applications of CLEC Technology--Laboratory Reactions
CLECs may be used as laboratory reagents in small columns or in batch
processes, which can be used to carry out laboratory reactions. Some of
the broad categories of reactions are in Table 5. In addition,
appropriately selected antibodies, or antibody fragments (particularly
monoclonal antibodies), which recognize process chemicals, clinical
analytes, pesticides and other environmental residues can be turned into
CLACs and used to selectively bind these substances, making their
detection in and/or removal from a sample or other source possible. For
example, a substance can be separated from a mixture by contacting the
mixture with a crosslinked immobilized antibody crystal in which the
antibody recognizes the substance, thereby producing a complex of the
substance and the crystal, and separating from the mixture the resulting
complex. The crosslinked crystal will generally be linked to a solid
support, such as a column or a bead. For example, CLACs can be packed into
an affinity column and a sample from which a selected component is to be
removed can be run through the column resulting in binding of the
component to the antibody and permitting its removal. This can be used,
for example, on a small or a large scale to remove pesticides (e.g.,
Aldrin) from soil, water or other source. In this case, a pesticide
binding monoclonal antibody CLAC is used in an affinity column or other
appropriate structure. Because it is in CLAC form, the monoclonal antibody
can withstand the harsh conditions used (e.g., organic solvents) and the
antibody-bound pesticide is removed from the sample (e.g., by nature of
the fact it is bound to antibody which is, itself, bound to a solid
support, which can be separated from the sample.
TABLE 5
__________________________________________________________________________
Enzyme Employed
Reaction type catalyzed
Reference
__________________________________________________________________________
lipases, phospholipases
Stereoselective synthesis: including
Zaks, A. & Klibanov, A. M.
esterification, transesterification
Proc. Nat. Acad. Sci. USA.
aminolysis, lactonizations
82: 3192-3196 (1985)
polycondensations, acylation,
Klibanov, A. M. Acc. Chem. Res.
oximolysis and resolution of racemic
23: 114-120 (1990) and references therein
mixtures Wong, C. H., Chemtracts-Organic
Chemistry 3: 91-111 (1990) and
references therein
esterases stereoselective synthesis and
Kobayashi et al., Tetrahedron Letters
resolution Vol 25, #24: 2557-2560 (1984)
Schneider et al., Agnew. Chem. Int.
Ed. Engl. 23 (#1): 64-68 (1984)
tyrosinase oxidation of phenols to produse
Kazandjian, R. Z and Klibanov, A. M.
quinones J. Am. Chem. Soc 110: 584-589 (1986)
protease, eg subtilisin
stereoselective acylation of
Riva et al. J. Am. Chem. Soc.
carbohydrates 110: 584-589 (1988)
oxidases selective oxidation of hydrocarbons
Klibanov, A. M. Acc. Chem. Res.
23: 114-120 (1990) and references therein
other enzymes not
Stereoselective synthesis:
Wong, C. H., Chemtracts-Organic
requiring co-factors: Chemistry 3: 91-111 (1990) and
isomerases, lyases, references therein
aldolases, glycosyl
transferases, glycosidases
other enzymes not
Stereoselective synthesis:
Wong, C. H., Chemtracts-Organic
requiring added co-factors: Chemistry 3: 91-111 (1990) and
flavoenzymes, pyridoxal references therein
phosphate enzymes,
metalloenzymes
enzymes requiring co-
Stereoselective synthesis:
Wong, C. H., Chemtracts-Organic
factors: kinases (ATP), Chemistry 3: 91-111 (1990) and
oxidoreductases (NAD/P), references therein
methyl transferases
(SAM), CoA-requiring
enzymes, sulfurases
(PAPS)
__________________________________________________________________________
Schneider et al. (Agnew. Chem. Int. Ed. Engl. 23 (No.1): 64-68 (1984))
illustrates how enzymes may be used in organic syntheses. Pig liver
esterase was employed in the meso-ester transformation into a chiral
mono-ester in an aqueous phosphate buffer.
The advantages of CLEC catalyzed reactions for laboratory use are
threefold. First, CLECs retain high activity in harsh environments (eg.
aqueous, organic, near-anhydrous solvents and mixtures of these, and at
high temperatures) that are typical of laboratory chemical synthesis
experiments. Second, CLECs exhibit high operational and storage stability,
which is appropriate for intermittent laboratory experiments. Third, their
high activity per unit volume will allow shorter reaction times and
require smaller volumes of enzyme (per unit of activity). Thus, the
advantages that CLECs offer over free or immobilized enzymes, provide
organic chemists with an alternative, highly selective, synthetic tool.
Enzymes are catalytic proteins that make possible or greatly accelerate
almost every biologically important chemical reaction. Enzymes perform
oxidations, reductions, additions, eliminations, rearrangements,
hydrolyses, dehydrations. They are thus responsible for manufacturing
every biologically significant molecule in plants and animals, from
sub-cellular organelles to bones and organ systems. Because an enzyme
typically carries out one chemical step at a time in what may be a
complex, multistep transformation (requiring twenty or more enzymes),
chemists have for decades been intrigued by the potential for using
enzymes as synthetic chemical catalysts.
Despite the great potential for using enzymes in synthetic chemistry, their
utility outside living systems has been hampered by seemingly
insurmountable problems:
Isolation and purification of enzymes is difficult
Enzymes are susceptible to degradation or inactivation by air, changes in
pH, other enzymes, and organic compounds such as solvents
The cost of enzymes and their chemical cofactors (small molecules required
for enzymatic action) is high
Relatively little is known about the interactions between enzymes and
organic molecules which are not their natural substrates
Fortunately, biotechnology is beginning to solve problems associated with
isolation, purification, and cofactor regeneration, and innovative
chemists are compiling data almost daily on adapting known enzymes to new
substrates. However, the instability of enzymes and their aversion to
solvents other than water have remained barriers to widespread use of
these proteins in routine organic synthesis.
A CLEC (cross-linked enzyme crystal) contains more than one protein
molecule organized in a crystalline lattice, which is either crosslinked
or not crosslinked, or crosslinked by simple bifunctional organic
molecules. A typical crosslinking agent, glutaraldehyde, contains two
sites for attaching free amino groups found on proteins. As long as the
amino group that attaches to glutaraldehyde is far from the enzyme's
active site, crosslinking has no appreciable effect on enzymatic activity.
After forming, the CLEC may be freeze-dried, air dried or left in a liquid
state. In any of these storage conditions they may be stored indefinitely
as a solid at room temperature. But most importantly, CLECs retain their
high activity in real-world chemical reaction conditions, including harsh
temperatures and pH, in many types of organic or mixed aqueous/organic
solvent, and even in the presence of other enzymes that digest proteins.
Since so many industrial applications of enzymes depend on the molecule's
stability and activity in sub-optimal conditions, CLECs will greatly
expand the use of industrial and research enzymes, as well as non-enzyme
proteins and a wide range of peptides. CLECs replace many conventional
enzymes in applications where soluble or immobilized enzymes are already
used, as well as in new applications where they cannot be used, e.g., in
organic solvents or in mixed solutions of water and non-polar or polar
organics, such as acetone, dioxane, acetonitrile, and tetrahydrofuran.
Some potential chemical uses of CLECs in industry and research include:
Organic synthesis of important high-value intermediates and specialty
chemicals.
Chiral synthesis and resolution for optically pure pharmaceuticals and
specialty chemicals.
Bioprocessing of products produced through fermentation, as well as natural
products harvested from plants, animals, and insects.
But CLEC technology is not just for enzymes. It is a general method for
achieving unprecedented stability and activity in almost any protein and
in smaller peptides. The most exciting applications of CLEC technology may
arise not from chemical catalysis in the laboratory, but in therapeutics
and diagnostics--both enzymatic and non-enzymatic--in the human body,
tissues, or fluids.
There is considerable interest among pharmaceutical companies in proteins,
protein fragments, and synthetic peptides as drugs and diagnostics. More
and more drugs in research and development are peptides or "peptide-like"
synthetic organic compounds. The trouble with peptides and peptide-like
compounds--whether they are natural or synthetic--is stability. The human
body contains thousands of proteolytic enzymes (proteases) that break down
proteins and peptides into smaller and smaller units. Because of
degradation by proteases, some peptides with potential therapeutic or
diagnostic benefit have biological half-lives that are too short to enable
them to work. Pharmaceutical scientists try to circumvent degradation of
synthetic or semi-synthetic peptides by masking them with chemical groups
that confer some protection from proteases. Unfortunately, because of
strict requirements for chemical structure and molecular shape in
biochemical interactions, the use of chemical protecting groups tends to
lower a drug's affinity for target molecules or receptors.
Applied to non-enzymatic peptides and proteins, CLEC technology can produce
therapeutic and diagnostic compounds with unprecedented in vivo stability
and near-theoretical activity. Crosslinking may allow drug researchers to
use native proteins or peptides and may eliminate the need, in certain
instances, for designing structural analogues or implementing masking
strategies to make drugs more resistant to degradation. Some potential
medical uses of CLECs (or CLEC technology) include:
Therapeutic antibodies that bind to and inactivate viruses, bacteria, or
proteins; antibodies against inflammatory mediators and mediators of nerve
and tissue destruction; antibodies to messenger chemicals in conditions
such as Alzheimer's disease, stroke, nervous system trauma, and autoimmune
diseases; catalytic antibodies, which react with their immunologic targets
after binding to them.
Diagnostic antibodies that bind to their targets to allow detection in
vitro or in vivo.
Therapeutic proteins and enzymes that replace proteins absent due to acute,
chronic, or inherited diseases; enzymes that dissolve blood clots or
cholesterol deposits.
"Super-Inhibitors" of enzymes, receptors, or small molecules made from
fragments of naturally-occurring inhibitor molecules or even from
synthetic peptides.
Radiology: Proteins or peptides connected to radioactive elements for
medical diagnostics (imaging) and therapeutics (radiotherapy); combined
radiotherapy/imaging.
Enzyme therapy for cancer nutrient deprivation, pancreatic insufficiency,
and other enzyme or protein therapies which, by the use of an enzyme or a
protein results in the elimination of an adverse health state, and
extracorporeal treatment (in which blood, lymph, or other tissues are
removed from the patient, treated, and re-introduced)
Oral peptide drug delivery--conventional peptide drugs are quickly broken
down in the gut, mostly by proteases. The improved stability of CLECs
toward proteolysis may make these compounds attractive alternatives to
intravenously administered peptide and peptide-like drugs.
Other important applications of CLECs include biosensors. Biosensors
include many types of devices and technologies that detect and quantify
biologically important events. For our purposes, however, biosensors are
immobilized molecules connected to some type of signal transducer--either
optical, electrical, electromagnetic, or chemical--that produce a signal
in the presence of an analyte biomolecule. Many biosensors simply detect
the presence (or absence) of an analyte. More sophisticated sensors also
quantify the amount of the analyte. Commercially available antibody-based
biosensors are based on antibody-antigen interactions, one of the most
specific chemical interactions known between two molecules.
Antibody-based biosensors have the same problems as other technologies
based on purified enzymes: reduced operational and storage stability,
vulnerability to contamination, and susceptibility to degradation when
used in living tissues or on bodily fluids. Other technical hurdles to
enzyme biosensors include difficulty in miniaturization, integration of a
biomolecule and transducer, contamination and cross-reactivity.
CLEC technology for biosensors and diagnostics is applicable to enzyme
antibodies, catalytic antibodies and other reaction proteins. These
products may include:
Agricultural testing for pesticides, parasites, toxins, drug residues, and
food composition.
Environmental monitoring and control
Medical and veterinary diagnostic tests for home, clinics, hospitals,
clinical laboratories, and physician offices.
Industrial process monitoring, primarily in biotechnology
Real-time monitoring and sensing for research and therapy using animals,
tissues, or cells.
CLECs offer many advantages over non-crosslinked enzymes. These advantages
also hold for non-enzymatic proteins, and perhaps for smaller peptides as
well:
Superior Stability--The intermolecular contacts and crosslinks between
enzymes in the crystal lattice of a CLEC stabilize the enzyme and prevent
denaturation. CLECs remain active and are resistant to proteolysis,
extremes of temperature, and organic solvents. CLECs are stable at room
temperature indefinitely.
Solid Form--Soluble enzymes require immobilization in most applications.
CLECs, which can be either soluble or insoluble enzyme particles,
eliminate the need for an inert support.
Highest Possible Concentration--the activity, per unit volume, of CLECs is
significantly higher than that of conventionally immobilized enzymes or
concentrated soluble enzymes. Enzyme concentrations within a CLEC are
close to theoretical limits.
Superior Uniformity--As crystals, individual CLEC particles are uniform and
monodisperse, in contrast to undefined, and often random attachment of
conventional enzymes. CLECs remain monodisperse on reconstitution, even in
organic solvents.
Operational Convenience--CLECs can easily be freeze-dried or air dried and,
in that form, can be stored indefinitely at room temperature.
CLECs have special advantages over soluble or conventionally immobilized
enzymes for bioprocessing, including:
Improved yield under harsh conditions or situations requiring high
throughput, enabling process chemists to concentrate on maximizing yield
with less concern about reaction conditions.
Cleaner, faster reaction--Because they are not soluble, CLEC enzymes can be
easily separated from the product via settling or filtration, thereby
eliminating a source of contamination.
Longer catalyst life and faster reaction times.
Environmentally benign--biocatalysts are easier to dispose of than most
synthetic catalysts.
Mechanical strength--allowing continuous flow rather than batch processes.
As biosensors, CLECs have all the advantages of immobilized enzymes and
none of the drawbacks:
High specificity, sensitivity, and accuracy.
Extended operational lifetime and storage stability.
Miniaturization--Since CLECs are insoluble crystals, they do not require an
inert carrier and exhibit the highest protein density possible. The high
specific activity means a CLEC will generate the largest possible signal
from even the smallest substrate (analyte) concentration.
Uniform Signal--Proteins in crystal form are uniformly arranged. This
uniformity should produce a linear and predictable signal.
Resistance to Contamination--Soluble enzymes are vulnerable to proteolysis
as well as contamination in many biosensor environments. Since CLECs are
crystalline they are less susceptible to contamination and may be reused
with minimum effort.
CLECs will have a primary impact on companies involved in industrial and
research enzymes, medical therapeutics and diagnostics, and biosensors.
These firms will now be able to direct their research efforts toward
producing biocatalysts, drugs, and other products with less concern for
the stability of their molecules and more concern for function.
Most CLECs retain nearly 100% of the activity of soluble enzymes after two
weeks or more in conditions that denature soluble proteins within hours.
Improvements in efficiency and throughput per weight of protein can
therefore easily reach a hundred-fold or more. That may mean, in the short
term, selling less of an enzyme or antibody compared to the soluble form.
However, the improved performance of CLECs may expand the commercial
potential for proteins as much as ten-fold for existing applications, and
thousands of times for as-yet undiscovered uses.
CLEC technology will cause pharmaceutical and specialty chemical
manufacturers to re-think the catalytic potential of many enzymes
processes (and non-enzyme proteins and peptides) that may have been
shelved due to the low efficiency of idealized soluble or supported
proteins, the high cost of multi-phase reactions, or difficulties in
product isolation or biocatalyst removal.
Pharmaceutical companies concerned about the in vivo proteolysis or
unacceptable biological half-life of protein or peptide therapeutics can
now focus on pharmacologic activity rather than on masking peptide bonds
from circulating degrading enzymes.
Of course, some proteins may not have free primary amine groups to combine
with glutaraldehyde. In others, the free amines may be too close to the
active site or may be required to retain tertiary structure necessary for
activity. So although the prototype CLEC uses glutaraldehyde conjugated
with primary amines, this approach may not be appropriate to every
protein, enzyme, antibody, or peptide. For these molecules, other suitable
crosslinkers can be employed.
In general, CLECs will greatly expand the uses of industrial proteins,
especially enzymes and antibodies. It is difficult to pinpoint, from this
vantage point, the improvement in efficiency required to make a
run-of-the-mill peptide interesting scientifically. Predicting which
molecules will turn a profit is even more difficult, especially in highly
regulated healthcare fields. However, with hundred-fold improvements in
protein stability easily attainable via crosslinking, CLECs will resurrect
thousands of peptide research programs and greatly expand the scope of
currently successful research efforts.
Computer databases of proteins and peptides might lead to a nearly
limitless supply of leads for crosslinking, resulting in dozens or
hundreds of new products. Modifying refractory proteins through residues
containing sulfhydryl or hydroxyl groups might be another way to crosslink
proteins that are poor candidates for joining through free amine groups.
Other applications can include, for example, deposition of crystallized
enzymes, proteins, peptides, or amino acids on an inert substrate. Also,
coating can be formed over crystallized amino acid chains by crosslinking
any outer portion of the crystals with a suitable crosslinking agent, such
as an aldehyde or an oligomer of the aldehyde. An example of a suitable
aldehyde is glutaraldehyde.
In all of these instances described above, but not limited to these, the
method of this invention can be adapted by one of ordinary skill in the
art, to convert a process using a conventionally immobilized enzyme
catalyst to the use of a CLEC of the appropriate enzyme. CLECs can not
only replace conventional immobilized enzymes, but can also be used in
cell mediated transformations.
The present invention will now be illustrated by the following Examples,
which are not intended to be limiting in any way.
EXAMPLE 1
Crystallization and Crosslinking of Thermolysin for Synthesis of the
Aspartame Precursor, Z-Asp-Phe-OMe
Crystallization
250 mg of thermolysin from Bacillus thermoproteolyticus was purchased from
Boehringer-Mannheim GmbH, and dissolved in 4 ml of 45% dimethyl sulfoxide
(DMSO) and 55% 1.40 M calcium acetate, 0.50 M sodium cacodylate at pH 6.5.
These starting conditions are similar to those described by Matthews et.
al. for the production of diffraction quality thermolysin crystals (see,
eg., Holmes and Matthews, J. Mol. Biol. 160: 623-639 (1982)). The protein
solution was then concentrated to 1 ml in a Centricon 10
micro-concentrator. A good yield of microcrystals was obtained by a
process of flash crystallization, now disclosed here, in which 1 ml of
water, or 1.40 M calcium acetate, 0.50 M sodium cacodylate at pH 6.5, was
rapidly injected into either of the thermolysin-DMSO solutions described
above. A shower of hexagonal micro-crystals of approximately uniform
dimensions (approx. 10.sup.-1 mm in length) results from this process.
Crosslinking of Thermolysin Microcrystals
The protocol used in this specific example of the method of this invention
is an adaptation of that described by Nakanishi et. al. (Biotechnology 3:
459-464 (1985)), in which protocol, thermolysin was first adsorbed onto a
carrier bead composed of the ion-exchange resin Amberlite XAD-7, and
subsequently immobilized by cross-linking with glutaraldehyde (Quiocho and
Richards, Proc. Natl. Acad. Sci. (USA) 52:833-839 (1964)). In this
exemplification, the microcrystals of thermolysin obtained above were
centrifuged and pelleted, and the supernatant was discarded. 5 ml of 17.5%
technical grade glutaraldehyde, in 2.5% DMSO, 0.05M calcium acetate, and
0.025M sodium cacodylate at pH 6.5, were then added to the microcrystals.
The mixture was incubated with gentle agitation at 37.degree. C. for 4
hours. The crosslinking reaction was stopped by repeated washing of the
crystals with 10 ml aliquots of water to remove the glutaraldehyde
solution. The washed cross-linked thermolysin crystals constitute the
thermolysin CLEC used below as a catalyst.
Synthesis of Z-Asp-Phe-OMe in an Aqueous Solution
5 ml of a thermolysin CLEC suspension were added to a continuous
stirred-batch reactor incubated at 37.degree. C. After centrifugation and
decantation of the supernatant, an aqueous reaction mixture was added to
the CLECs. This solution was prepared by mixing 80 mg of Z-L-Asp and 80 mg
of L-Phe-OMe-HCl in 1 ml of water, with acetic acid added to obtain a pH
of 7.0. Samples were taken for analysis by HPLC. Table 6 below shows the
HPLC peak height of the Z-L-Asp substrate peak after the indicated time of
reaction, normalized to 1 at time t=0. Since Z-L-Asp is rate limiting in
this reaction, measuring its depletion is equivalent to measuring the
appearance of product Z-L-Asp-L-Phe-OMe (Nakanishi et. al. Biotechnology
3: 459-464 (1985)). Table 6 also includes the normalized peak height of
limiting Z-L-Asp substrate remaining, and an estimate of the degree of
completion of the reaction. It is clear that the reaction proceeded to
about 20% completion within the first 30 seconds and plateaued there.
These results are consistent with the observations of Nakanishi et al.
(Biotechnology 3: 459-464 (1985)) when using conventionally immobilized
thermolysin in an aqueous reaction mixture as above, and are attributable
to the sparing solubility of the Z-L-Asp-L-Phe-OMe product in water.
TABLE 6
______________________________________
Reaction
Time Peak Height
Percent
(sec) (Normalized)
Completion
______________________________________
0 1.000
30 0.727 27.3%
60 0.857 14.3%
120 0.940 6.0%
180 0.797 20.3%
______________________________________
Synthesis of Z-Asp-Phe-OMe in a Near-anhydrous Solution
5 ml of a thermolysin CLEC suspension were added to a continuous
stirred-batch reactor incubated at 37.degree. C. After centrifugation and
decantation of the supernatant, a near-anhydrous organic reaction mixture
was added to the CLECs. This solution was prepared by mixing 80 mg of
Z-L-Asp and 240 mg of L-Phe-OMe in 1 ml of 99% ethyl acetate and 1% water.
Samples were taken for analysis by HPLC. Table 7 below shows the HPLC peak
height of the Z-L-Asp substrate peak after the indicated time of reaction,
normalized to 1 at time t=0. Since Z-L-Asp is rate limiting in this
reaction, measuring its depletion is equivalent to measuring the
appearance of product Z-L-Asp-L-Phe-OMe (Nakanishi et. al. Biotechnology
3: 459-464 (1985)). Table #7 also includes the normalized peak height of
limiting Z-L-Asp substrate remaining, and an estimate of the degree of
completion of the reaction. In this case, the reaction proceeded to about
70% completion within the first 30 seconds and plateaued there. These
results are also consistent with the observations of Nakanishi et al.
(Biotechnology 3: 459-464 (1985)) with conventionally immobilized
thermolysin in a near-anhydrous reaction mixture, and are attributable to
product inhibition of the enzyme.
TABLE 7
______________________________________
Reaction
Time Peak Height
Percent
(sec) (Normalized)
Completion
______________________________________
0 1.000
30 0.323 67.7%
60 0.314 68.6%
120 0.305 69.5%
180 0.272 72.8%
______________________________________
EXAMPLE 2
Crystallization, Cross-linking and Lyophilization of Thermolysin and
Assessment of Characteristics of Resulting Product
Crystallization of Thermolysin
Thermolysin (Diawa Kasei K. K., Japan) was dissolved in 10 mM calcium
acetate (Sigma), pH 10.0, to a concentration of 10% (w/v). The pH of the
solution was maintained at 10.0 by titration with 2 M NaOH. Following
complete solubilization, the protein solution was titrated to pH 8.0 with
2 M HCl. Solid calcium acetate was added to 1.2 M. Dimethyl sulfoxide
(Sigma) was then added to 30%. The protein was concentrated to 100 mg/ml
by ultrafiltration in an Amicon stir cell (10,000 MWCO membrane).
Concentrated enzyme was aliquoted and stored at -70.degree. C. Thermolysin
was crystallized by the addition of 9 volumes demineralized water to 1
volume concentrated (100 mg/ml) protein solution. The solution was briefly
vortexed and allowed to stand overnight at room temperature. Crystals were
washed with 10 volumes of 10 Mm calcium acetate pH 7.0 and recovered by
low speed centrifugation (10 min at 1500.times.G, Beckman GPR centrifuge).
The rapid addition of water to a concentrated (100 mg/ml) solution of
thermolysin induces the formation of crystals which become visible under
low-power magnification within ten minutes. Crystal size is reproducibly
dependent on the final protein concentration. Three volumes of water to
one volume of thermolysin concentrate (100 mg/ml) will produce 0.Smm long,
X-ray diffraction quality hexagonal rods that correspond to the crystals
described earlier by Colman et al. (Colman, P. M., Jansonius, J. N. and
Matthews, B. W., J. Mol. Biol. 70: 701-724 (1972)), as confirmed by us by
diffraction analysis. Adding ten volumes of water to one of protein
concentrate reduces the length of the resulting crystals to 0.05 mm. These
micro-crystals are preferred in CLEC applications, since they tend to
minimize diffusion problems related to crystal size (see eg., Quiocho, F.
A. and Richards, F. M. Biochemistry 5: 4062-4076 (1967)). Within a given
batch of protein, crystal size was consistently uniform. (Crystals
0.05-0.10 mm in length were used in this study to facilitate accurate
pipetting of crystalline suspensions.) Densitometer scans of SDS-PAGE
showed a six-fold purification of the enzyme on crystallization,
significantly increasing the specific activity of the CLECS.
Crystallization resulted in a 20% decrease in the total activity of the
CLEC protein compared to soluble thermolysin, when assayed by
spectrophotometric cleavage of the dipeptide substrate
furylacryloyl-glycyl-L-leucine-amide (FAGLA), as described below.
Crosslinking of Thermolysin Crystals
Thermolysin crystals were crosslinked for 3 hours at room temperature in a
solution of 12.5% glutaraldehyde (Sigma), 5% DMSO and 50 mM Tris pH 6.5.
The crosslinked crystals were washed 3 times in demineralized water and
recovered by low speed centrifugation, as described in respect to
crystallization of thermolysin. Chemical cross-linking of enzyme crystals
stabilizes the crystal lattice and the constituent enzyme molecules in the
crystal sufficiently so as to permit the practical use of CLECs in
environments that are otherwise incompatible with enzyme function. There
was no measurable difference in enzymatic activity between the crosslinked
and un-crosslinked crystals when assayed (spectrophotometrically) by
monitoring cleavage of the dipeptide substrate FAGLA (described below).
Moreover, cross-linking stabilizes CLECs to the point that they can be
lyophilized, with retention of full enzymatic activity upon reconstitution
in aqueous, organic, and mixed aqueous-organic solvents as shown in FIG. 1
and Table 8. Although crystallization resulted in a 30% decrease in the
specific activity of the CLEC protein compared to soluble thermolysin,
crosslinking and lyophilization of the CLECs did not further diminish
specific activity.
TABLE 8
______________________________________
Thermolysin Activity
Time Absorbance 345 nm
(min) CLEC Soluble Enzyme
______________________________________
1 0.0 0.314 0.315
2 1.0 0.272 0.271
3 3.0 0.235 0.218
4 5.0 0.204 0.198
5 10.0 0.184 0.185
6 15.0 0.183 0.184
______________________________________
Enzymatic Activity of Soluble and CLEC Thermolysin
The catalytic activity of soluble and CLEC thermolysin was assayed (Feder,
J. and Schuck, J. M., Biochemistry 9: 2784-2791 (1970)) by hydrolysis of
the blocked dipeptide substrate furylacryloyl-glycyl-L-leucine-amide
(FAGLA) (Schweizerhall). Cleavage of the amide bond was measured
spectrophotometrically by a decrease in absorbance at 345 nm. Initial
enzyme concentration was 10.sup.-7 M by Bradford protein determination and
densitometer scanning (Pharmacia LKB UltroScan XL) of Coomassie stained
SDS-PAGE gels. CLEC enzyme is defined as reconstituted lyophilized
crosslinked thermolysin crystals. Soluble enzyme is defined as thermolysin
concentrated to 100 mg/ml. Enzyme was added to a 5 ml reaction volume
containing substrate. Aliquots of the reaction mix were removed at the
indicated times, and absorbance at 345 nm was measured. CLEC thermolysin
was separated from the reaction mix by brief centrifugation (Beckman,
microcentrifuge E) before reading absorbance. Absorbance was fitted to a
pseudo first order rate equation and kcat/Km was calculated by dividing
the fitted value by enzyme concentration (Multifit 2.0 Curve Fitting for
the Apple Macintosh Computer, Day Computing P.O. Box 327, Milton,
Cambridge CB4 6WL, U.K. (1990)).
pH Dependence and Stability
The pH optimum and stability of the soluble enzyme were compared to that of
thermolysin CLECs by cleavage of the dipeptide substrate FAGLA. Results
are shown in FIG. 2 and Table 9. Both soluble and crystalline enzyme forms
demonstrate maximum activity at pH 7. CLECs and soluble thermolysin also
demonstrated similar activity in the acidic range and the bell shaped pH
profile generated by the soluble enzyme was in good agreement with
published data (Feder, J. and Schuck, J. M., Biochemistry 9: 2784-2791
(1970)). In the alkaline pH range, however, the crystalline enzyme
maintains maximum activity, to pH 10, while the soluble enzyme has 75%
activity at pH 8.5, and only 25% activity at pH 9. At pH 9.5, the soluble
enzyme is completely inactive.
TABLE 9
______________________________________
Thermolysin pH Curve
% Maximum Activity
pH CLEC Soluble Enzyme
______________________________________
1 5.0 10.250 5.170
2 5.5 9.750 6.070
3 6.0 52.500 39.100
4 6.5 85.000 74.610
5 7.0 97.500 100.000
6 7.5 100.000 98.650
7 8.0 97.500 82.920
8 8.5 95.000 71.910
9 9.0 96.250 24.720
10 9.5 95.000 0.000
11 10.0 90.000 0.000
______________________________________
Stability at Elevated Temperature
One can achieve higher reaction rates and lower diffusion times for
substrates and products by operating a given chemical process at higher
temperature, where one is usually limited by the temperature stability of
substrates and products. In enzyme-based catalysis, however, it is often
the loss of enzymatic activity that sets the practical limit on the
temperature that a process can be run. The additional stabilization
achieved in CLECs allows for enzymatic activity at much higher
temperatures than is possible for soluble enzyme.
The enhanced stability at lower temperatures simplifies the routine long
term storage of the CLEC catalysts. For example, it was necessary to store
concentrated (>50 mg/ml) solutions of soluble thermolysin at -80.degree.
C. to retain maximum specific activity. At room temperature, activity was
usually lost within one day. In contrast, rehydrated thermolysin CLECs
could be routinely stored for months at room temperature with no apparent
loss of activity. Unreconstituted lyophilized CLECs of thermolysin appear
to be viable indefinitely.
Thermal stability and resistance to autolysis were demonstrated in
thermolysin CLECs following incubation at 65.degree. C. for five
consecutive days (FIG. 3 and Table 10). Thermolysin CLECs retained maximum
activity after five days incubation at elevated temperature. In contrast,
the soluble thermolysin lost 50% of its initial activity after only two
hours incubation and demonstrated negligible activity after 24 hours
incubation at 65.degree. C.
TABLE 10
______________________________________
Thermolysin Thermal Stability at 65.degree. C.
Time % Maximum Activity
(days) CLEC Soluble Enzyme
______________________________________
1 0.000 100.000 100.000
2 0.041 70.000
3 0.083 96.000 50.000
4 0.164 32.000
5 0.246 17.000
6 0.410 97.0 10.000
7 1.000 101.0 2.000
8 2.000 97.0
9 3.000 94.0
10 4.000 96.0
11 5.000 92.0
______________________________________
The activity of soluble and CLEC thermolysin was measured following
incubation at 65.degree. C. Soluble thermolysin was incubated in 10 mM
calcium acetate, 50 mM Tris pH 7.0 in a 65.degree. C. water bath. The
reaction volume was 500 .mu.l. Final protein concentration was 10 mg/ml.
Aliquots were removed at times 0, 1, 2, 4, 6, 10, and 18 hours. The
samples were assayed by SDS-PAGE and FAGLA cleavage at room temperature as
described above. For the thermolysin CLECs, a 250 .mu.l crystal suspension
in 10 mM calcium acetate and 50 mM Tris was also incubated in a 65.degree.
C. water bath. Activity was assayed at times 0, 1, 6, 24, 48, 72, 96, and
120 hours by FAGLA cleavage.
Resistance to Exogenous Proteolysis
Assessment of the resistance of the thermolysin CLEC to the action of an
exogenous protease was also carried out. SDS-PAGE (Sodium dodecyl sulfate
poly acrylamide gel electrophoresis) analysis suggests that commercial
enzymes can contain a substantial percentage of contaminants, some of
which might have proteolytic activity against the principal soluble enzyme
species. Given the packing of enzyme molecules in a crystal lattice one
might assume that the interior enzyme molecules in a CLEC would be
protected from proteolysis. To test this possibility, thermolysin CLECs
and a soluble enzyme preparation were incubated in the presence of the
streptococcal protease, Pronase.RTM., a nonspecific protease capable of
digesting most proteins to free amino acids (Calbiochem 1990 Catalog;
LaJolla, Calif.).
Soluble and CLEC thermolysin were incubated in 50 mM Tris, pH 7.5, at
40.degree. C. in the presence of the protease Pronase.RTM. (Calbiochem).
The Pronases to thermolysin ratio was 1/40. To inhibit thermolysin
autolysis and prevent the proteolytic destruction of pronase by the
thermolysin, EDTA was added to the soluble enzyme reaction to a final
concentration of 100 mM (EDTA inhibits thermolysin activity but not
Pronase.RTM.). At the times indicated aliquots were removed from the
reaction mix and activity was assayed spectrophotometrically by cleavage
of the dipeptide substrates FAGLA. To offset thermolysin inhibition due to
the presence of EDTA, the spectrophotometric assay of soluble enzyme
activity was performed in 0.5 M calcium acetate buffer pH 7.0 and enzyme
concentration was increased two fold. Crosslinked crystalline enzyme was
assayed as described above.
As can be seen in FIG. 4 and Table 11, the soluble thermolysin was rapidly
degraded and lost all activity after 90 minutes incubation. In contrast,
the activity of the thermolysin CLEC was unaffected by four days
incubation in the presence of protease. This near imperviousness to
proteolysis is of particular interest in diagnostic biosensor applications
where a suitable CLEC might be called upon to act in the presence of an
unknown cocktail of naturally occurring proteolytic enzymes.
TABLE 11
______________________________________
Protease Resistance
Time % Maximum Activity
Time
(days) CLEC Soluble Enzyme
(min)
______________________________________
1 0.000 100.0 100.0 0.000
2 0.003 25.0 5.000
3 0.010 17.5 15.000
4 0.021 9.5 30.000
5 0.042 98.0 3.0 60.000
6 0.063 1.0 90.000
7 0.084 101.0 0.0
8 1.000 97.0
9 2.000 99.0
10 3.000 98.0
11 4.000 96.0
______________________________________
Stability in the Presence of Organic Solvent
In order for enzymes to gain ideal acceptance as viable industrial
catalysts, they must be able to function without excessive intervention in
the practical environment of manufacturing processes. In particular, this
would include the use of aqueous, polar and non-polar organic solvents,
and mixtures of these. In commercial applications, aqueous-organic solvent
mixtures allow manipulation of product formation by taking advantage of
relative solubilities of products and substrates.
Soluble thermolysin and thermolysin CLECs exhibited markedly different
stability in the presence of organic solvents. (Table 12). Soluble enzyme
concentrations which could be incubated in organic solvent were limited to
a maximum of 10 mg/ml. Concentrations greater than this value resulted in
the instantaneous precipitation of thermolysin upon addition of organic
solvent. In contrast, thermolysin CLEC concentrations were limited only by
the volume occupied by the crystals. Soluble thermolysin retained the
greatest activity (75%) following incubation in acetone, and the least
(36%) in tetrahydrofuran. Following a one hour incubation in the presence
of acetonitrile or dioxane the soluble enzyme lost approximately 50% of
its initial activity. The CLEC thermolysin retained greater than 95%
maximum activity following incubation with all organics assayed.
TABLE 12
______________________________________
% Maximum Activity
Soluble Enzyme
CLEC
______________________________________
Acetonitrile 42 102
Dioxane 66 97
Acetone 75 99
THF* 36 96
______________________________________
*Tetrahydro Furan
Stability in Organic Solvents
Thermolysin CLECs or soluble thermolysin preparations were incubated in 50%
(v/v) solutions of the indicated organic solvents. A 100 .mu.l slurry of
thermolysin CLECs (10 mg/ml) in 10 mM Tris pH 7 was placed in a 1/2 dram
glass vial. An equal volume of the indicated organic solvent was added and
the mixture was briefly vortexed. Twenty .mu.l of soluble thermolysin (100
mg/ml) was diluted in 80 .mu.l of 0.015M Tris buffer pH 7.0 in a 1/2 dram
glass vial. A 100 .mu.l volume of organic solvent was then added to the
protein solution and briefly vortexed. CLEC and soluble enzyme were
incubated in the presence of organic solvent for one hour at 40.degree. C.
Following incubation, enzyme activity was assayed by cleavage of the
dipeptide substrate FAGLA as described.
Low water concentration is thought to disfavor unfolding to intermediate
states on the path to enzyme denaturation. In CLECs, this restriction of
conformational mobility is provided by the inter-molecular contacts and
cross-links between the constituent enzyme molecules making up the crystal
lattice, rather than by the near-absence of water in the medium. As a
result, intermediate water-organic solvent concentrations are readily
tolerated by enzymes when formulated as CLECS, something previously
unobserved with enzymes (see Table 12). This discovery opens up whole new
areas of synthetic chemistry to exploitation using enzyme catalysis.
Even in near-anhydrous organic solvents, however, the routine use of
enzymes has been hampered by their tendency to form ill-defined
suspensions that are subject to clumping and other aggregation problems.
This property makes these preparations inherently unattractive for large
scale industrial processes. In contrast, CLECs and the constituent enzymes
within the crystal lattice, remain mono-disperse in all these solvents.
Comparison with Other Immobilization Methods
A number of useful reviews of enzyme immobilization methods have appeared
in the literature (Maugh, T. H., Science, 223: 474-476 (1984)); Tramper,
J., Trends in Biotechnology 3: 45-50 (1985)). In these, the enzyme always
represents a small fraction of the total volume of the immobilized
particle, the bulk of it being inert carrier material. The carrier
increases the mean free path between the solvent exterior of the
immobilized enzyme particle and the enzyme active sites, exacerbating
diffusion problems (Quiocho, F. A. and Richards, F. M., Biochemistry 5:
4062-4076 (1967)).
In a CLEC, the crosslinked crystal matrix provides its own support,
eliminating the need for a carrier. As a result, the concentration of
enzyme in a CLEC is close to the theoretical packing limit that can be
achieved for molecules of a given size, greatly exceeding densities
achievable even in concentrated solutions. The entire CLEC consists of
active enzyme, and thus, the diffusion-related reduction of enzyme
reaction rates usually observed with conventionally immobilized enzymes
relative to enzymes in solution are minimized (See FIG. 1), since the mean
free path for substrate and product between active enzyme and free solvent
will be greatly shortened for CLECs (compared to a conventional
immobilized enzyme carrier particles). Importantly, the constituent enzyme
in CLECs is intrinsically mono-disperse, and can be recovered by simple
manipulations of the CLEC particles, such as filtration, centrifugation or
decantation of solvent.
EXAMPLE 3
Soluble and CLEC Thermolysin-Catalysed Synthesis of the Aspartame Precursor
Z-Asp-Phe-OMe
Soluble and CLEC thermolysin catalysed synthesis of the aspartame precursor
was performed in a repeated batch experiment. Three different experimental
parameters were assessed: 1. thermolysin CLEC versus soluble thermolysin
half-life, 2. thermolysin CLEC versus soluble thermolysin specific
activity (equivalent protein concentration) and 3. thermolysin CLEC versus
soluble thermolysin total activity (equivalent protein dry weight).
The reagents were prepared as follows:
Solvent--a buffer (50 mM MES-NaOH, 5 mM CaCl.sub.2
(2-[N-morpholino]ethane-sulfonic acid)(Sigma) saturated ethyl acetate, pH
6.0) saturated solution of ethyl acetate (Nakanishi, K. et al.,
Biotechnology 3:459-464 (1985)). The MES buffer was prepared by dissolving
9.76 g MES and 0.102 g CaCl.sub.2 in 90 ml deionized water. The pH was
adjusted to 6.0 with 5 N NaOH. The volume was adjusted to 100 ml. To
prepare buffer saturated ethyl acetate, 10 ml MES buffer was combined with
90 ml ethyl acetate in separatory funnel, following agitation the organic
phase was collected.
Substrates-240 mM L-Phe-O-Me, 80 mM CBZ-L-Aspartic Acid. L-Phe-O-Me was
prepared by chloroform extraction of L-Phe-O-Me HCl. Equimolar amounts of
L-Phe-O-Me HCl and Na.sub.2 CO.sub.3 were dissolved in deionized water and
agitated with an appropriate amount of chloroform to extract the
L-Phe-O-Me. The chloroform was dehydrated with an appropriate amount of
MgSO.sub.4 and evaporated at 40.degree. C. L-PheOMe was stored as a 2.4 M
solution in ethyl acetate at -20.degree. C.
Thermolysin CLECS-0.216 mM thermolysin CLECs (7.5 mg/ml enzyme,
approximately 15 mg/ml dry weight). Procedure for 3 ml batch reaction
0.214 g of N-CBZ-L-Asp was dissolved in 9 ml buffer saturated ethyl
acetate. 1 ml 2.4 M Phe-O-Me stock as described above was added to the
CBZ-aspartic acid. Lyophilized CLECs or lyophilized soluble thermolysin
was added to 3 ml of the reaction mix containing buffered solvent and
substrates. The reaction was incubated at 55.degree. C. with agitation, pH
was maintained at 6.0. The CLECs remained insoluble and were removed from
the reaction by filtration and low speed centrifugation. The conversion of
substrates to product was monitored by removing 0.1 ml of reaction mix at
1 hr intervals for TLC or HPLC. The reaction volumes were scaled to 19 ml
for the 24 hour continuous batch reactions.
Assay of Aspartame Precursor Product
The progress of the reaction was followed by thin layer chromatography
(TLC) (Lindeberg, G, J. Chem. Education 64:1062-1064 (1987)) (mobilr
phsdr; 1:1:3 water:acetic acid:N-butanol). Solid phase; silica gel.
Visualize by UV at 245 nm and ninhydrin, and by high performance liquid
chromatography (HPLC) (Nakanishi, K. et al. Biotechnology 3:459-464
(1985); Oyama, K. et al., Meth. in Enzymology 136:503-516 (1984); Ooshima,
H. et al., Biotechnology Letters 7:789-792 (1985)). The reaction was
monitored at 214 and 280 n. One hundred per cent conversion to product is
defined as one hundred per cent conversion of CBZ-aspartic acid to
aspartame precursor.
Continuous Batch Synthesis of the Aspartame Precursor (Half-life)
Soluble and CLEC thermolysin catalysed synthesis of the aspartame precursor
was performed continuously for 10 days in a repeated 24 hour batch
experiment under the conditions described above. CLEC thermolysin
concentration was 15 mg/ml dry weight. Soluble thermolysin (Diawa 10%
protein) concentration was 37.5 mg/ml dry weight. Product was recovered
and fresh substrate was added to the reaction every 24 hr. Enzyme was
recovered from the reaction mix by centrifugation and filtration. The %
product was assayed by TLC and HPLC as described above (FIGS. 5A-5C).
Batch Synthesis Data (total activity)
______________________________________
% Product
Time (hours) CLECs Soluble
______________________________________
0 0 0
1 27 0
2 63 3
3 95 5
4 100 7
______________________________________
EXAMPLE 4
Crystallization, Cross-linking and Lyophilization of Elastase and
Assessment of Characteristics of the Resulting Product
Crystallization of Elastase
Lyophilized porcine pancreatic elastase (Serva) was dissolved in 0.1 M
sodium acetate pH 5.0 to a concentration of 5 mg/ml (w/v) at room
temperature. Rod shaped elastase crystals were visible within one minute
of the complete salvation of the protein. The crystallization solution was
transferred to 4.degree. C. and crystallization was completed overnight.
Crystals were recovered by centrifugation as previously described.
Cross-linking of Elastase Crystals
A 200 .mu.l volume of elastase crystals was added to a 1.3 ml solution of
5.77% glutaraldehyde and 1.5 M sodium acetate pH 5.0. The crystals were
crosslinked for one hour with mild agitation (stir plate). Following
cross-linking the crystals were washed with three 15 ml volumes of 0.2 M
Tris pH 8.0. The elastase CLEC was lyophilized as described in Example 2.
Enzymatic Activity of Soluble and CLEC Elastase
The catalytic activity of soluble and CLEC elastase was assayed
spectrophotometrically by measuring hydrolysis of the substrate
succinyl--(Ala).sub.3 p-nitroanilide (Bachem) [Bieth, et al. Biochem. Med
11:350-357 (1974)] (Table 13, FIG. 6). Cleavage was monitored by
increasing absorbance at 410 nm. Initial substrate concentration was
2.times.10.sup.-4. Enzyme concentration was 2.8.times.10.sup.-7 M. CLEC or
soluble enzyme was added to a 5 ml reaction volume containing substrate in
0.2 M Tris pH 8.0. As described previously, CLEC enzyme was removed from
the reaction mix prior to measuring absorbance.
TABLE 13
______________________________________
Elastase Activity
Absorbance 400 nm
Time (min) CLEC Soluble Enzyme
______________________________________
1 0.0 0.000 0.000
2 0.5 0.103 0.205
3 1.0 0.195 0.390
4 2.0 0.366 0.672
5 3.0 0.523 0.923
6 4.0 0.657 1.098
7 5.0 0.780 1.227
8 6.0 0.888 1.326
9 7.0 0.974 1.393
10 10.0 1.170 1.512
11 15.0 1.365 1.586
______________________________________
Resistance to Exogenous Proteolysis
Assessment of the resistance of the elastase CLEC to the action of protease
was also performed under identical conditions as described for thermolysin
(Example 2). Activity of the soluble and CLEC enzyme, following incubation
with protease, was assayed by hydrolysis of the nitroanilide substrate as
described above (Table 14 and FIG. 7).
TABLE 14
______________________________________
Elastase Resistance to Proteolysis
% Maximum Activity
Time CLEC Soluble Enzyme
______________________________________
1 0.0 100.0 100.0
2 10.0 53.0
3 20.0 32.0
4 30.0 101.0 18.0
5 45.0 11.0
6 60.0 102.0 8.0
7 120.0 101.0 3.0
8 180.0 103.0 2.0
______________________________________
EXAMPLE 5
Crystallization, Cross-linking and Lyophilization of Esterase and
Assessment of Characteristics of the Resulting Product
Crystallization of Esterase
As disclosed here, 30 mg/ml ammonium sulfate suspension of pig liver
esterase (Fluka) was dissolved in 0.25 M calcium acetate pH 5.6 at room
temperature. Esterase crystals were visible within several minutes
following addition of the calcium acetate solution. The crystallization
solution was allowed to stand at room temperature and crystallization was
completed overnight. Crystals were recovered by centrifugation as
previously described in Example 2.
Cross-linking of Esterase Crystals
As disclosed here, a 300 .mu.l volume of esterase crystals were added to a
5 ml solution of 12.5% glutaraldehyde and 0.5 M sodium acetate pH 5.5. The
crystals were crosslinked for one hour with mild agitation (stir plate).
Following cross-linking the crystals were washed with three 15 ml volumes
of 0.5 M calcium acetate pH 6.3. The esterase CLEC was lyophilized as
previously described in Example 2.
Enzymatic Activity of Soluble and CLEC Esterase
The catalytic activity of soluble and CLEC esterase was assayed
spectrophotometrically by monitoring hydrolysis of the substrate
p-nitrophenyl acetate (Fluka) (Table 15 and FIG. 8). Cleavage was
monitored by increasing absorbance at 400 nm. Initial substrate
concentration was 0.001%. Enzyme concentration was 1.times.10.sup.-8 m.
CLEC or soluble enzyme was added to a 5 ml reaction volume containing
substrate in 0.25 M calcium acetate pH 6.3. As described previously in
Example 2, CLEC enzyme was removed from the reaction mix by centrifugation
prior to measuring absorbance.
TABLE 15
______________________________________
Esterase Activity
Absorbance 400 nm
Time (min) CLEC Soluble Enzyme
______________________________________
1 0.0 0.000 0.000
2 0.5 0.770 0.252
3 1.0 0.128 0.297
4 2.0 0.208 0.337
5 3.0 0.260 0.346
6 5.0 0.324 0.353
7 7.0 0.353 0.359
8 10.0 0.369 0.368
______________________________________
Resistance to Exogenous Proteolysis
Assessment of the resistance of the esterase CLEC to the action of protease
was also performed under identical conditions as described for thermolysin
(Example 2). Activity of the soluble and CLEC enzyme, following incubation
with protease, was assayed by hydrolysis of the substrate p-nitrophenyl
acetate as described above (Table 16 and FIG. 9).
TABLE 16
______________________________________
Esterase Resistance to Proteolysis
% Maximum Activity
Time CLEC Soluble Enzyme
______________________________________
1 0.0 100.0 100.0
2 10.0 68.0
3 20.0 47.0
4 30.0 99.0 25.0
5 45.0 20.0
6 60.0 97.0 16.0
7 120.0 94.0 10.0
8 180.0 91.0 6.0
______________________________________
EXAMPLE 6
Crystallization, Cross-linking and Lyophilization of Geotrichum candidum
Lipase and Assessment of Characteristics of the Resulting Product
Crystallization of Lipase
As disclosed here, the enzyme lipase (Geotrichum (G.) candidum) was
crystallized by vapor diffusion from an aqueous solution of 20 mg/ml
protein in 50 mM Tris pH 7 containing 8% ammonium sulfate. Bipyrimidal
crystals were visible after 20 to 30 days incubation at room temperature.
Crystals were recovered by centrifugation, as previously described in
Example 2.
Cross-linking of Lipase Crystals
As disclosed here, lipase crystals were added to a solution of 12.5%
glutaraldehyde and 50 mM Tris pH 5.6. The crystals were crosslinked for
one hour. Following cross-linking the crystals were washed with three 15
ml volumes of 50 mM Tris pH 7.0. The lipase CLEC was lyophilized, as
previously described in Example 2.
Enzymatic Activity of Soluble and CLEC Lipase
The catalytic activity of soluble and CLEC lipase was assayed
spectrophotometrically by monitoring hydrolysis of the substrate
p-nitrophenyl acetate (Table 17 and FIG. 10). Cleavage was monitored by
increasing absorbance at 400 nm. Initial substrate concentration was
0.005%. Enzyme concentration was 1.5.times.10.sup.-8 M. CLEC or soluble
enzyme was added to a 5 ml reaction volume containing substrate in 0.2 M
Tris pH 7.0 at room temperature. As described previously in Example 2,
CLEC enzyme was removed from the reaction mix by centrifugation prior to
measuring absorbance.
TABLE 17
______________________________________
Liperase Activity
Absorbance 400 nm
Time (min) CLEC Soluble Enzyme
______________________________________
1 0.0 0.000 0.000
2 1.0 0.013 0.021
3 5.0 0.094 0.116
4 10.0 0.164 0.186
5 15.0 0.248 0.258
6 30.0 0.346 0.357
7 45.0 0.407 0.420
8 60.0 0.461 0.459
9 90.0 0.497 0.502
______________________________________
EXAMPLE 7
Crystallization, Cross-linking and Lyophilization of Lysozyme and
Assessment of Characteristics of the Resulting Product
Crystallization of Lysozyme
Following the method of Blake, C. C. F. et al., Nature 196:1173 (1962) 200
mg of lyophilized hen egg white lysozyme (Boehringer Mannheim) was
dissolved in 2.5 ml of 0.04 M sodium acetate buffer pH 4.7 at room
temperature. Following solvation of the protein, 2.5 ml of 10% sodium
chloride were added to the lysozyme solution dropwise with stirring. The
crystallization solution was allowed to stand overnight at room
temperature, and crystallization was completed in forty-eight hours.
Crystals were recovered by centrifugation, as previously described in
Example 2.
Cross-linking of Lysozyme Crystals
As described here, a 500 .mu.l volume of lysozyme crystals was added to 10
ml of 24% glutaraldehyde and 50 mM Tris pH 5.6 containing 20% sodium
chloride. The crystals were crosslinked for 20 minutes with mild agitation
(stir plate). Following cross-linking the crystals were washed with three
50 ml volumes of 20 mM calcium acetate and 50 mM potassium chloride pH
5.3. The lysozyme CLEC was lyophilized, as previously described in Example
2.
Enzymatic Activity of Soluble and CLEC Lysozyme
The catalytic activity of soluble and CLEC lysozyme was assayed by
measuring the rate of hydrolysis of the substrate 4-methylumbelliferyl
N-acetyl-chitrioside (Fluka) (Yang, Y. and Hamaguchi, K. J. Biochem.
8:1003-1014 (1980) (Table 18 and FIG. 11). The release of
4-methylumbelliferone was followed fluorimetrically (Perkin Elmer Model
LS-50). Initial substrate concentration was 1.42.times.10.sup.-3. Enzyme
concentration was 3.times.10.sup.-7. CLEC or soluble enzyme was added to a
2 ml reaction volume containing substrate in 20 mM calcium acetate and 50
mM potassium chloride pH 5.3 at 42.degree. C. The amount of
4-methylumelliferone was determined fluorimetrically by measuring
fluorescence intensities at 450 nm with excitation at 360 nm. Slit width
for both excitation and emission was 10 mm. As described previously in
Example 2, CLEC enzyme was removed from the reaction mix by centrifugation
prior to measuring fluorescence.
TABLE 18
______________________________________
Lysozyme Activity
Fluorescence
Time (min) CLEC Soluble Enzyme
______________________________________
1 0.000 0.000 0.000
2 10.000 4.400 18.900
3 30.000 10.500 29.400
4 60.000 27.500 44.800
5 90.000 33.800 51.700
6 120.000 45.900 59.800
______________________________________
EXAMPLE 8
Crystallization, Cross-linking and Lyophilization of Asparaginase and
Assessment of Characteristics of the Resulting Product
Crystallization of Asparaginase
As a modification of the procedure described by Grabner et al. [U.S. Pat.
No. 3,664,926 (1972)] 25 mg of lyophilized asparaginase (Worthington) were
dissolved in 500 .mu.l of 50 mM sodium phosphate buffer pH 7.2. The
solution was cooled to 4.degree. C. and the pH adjusted to 5.0 with 1 M
acetic acid. Cold (-20.degree. C.) ethanol was then added dropwise to the
asparaginase solution to a final concentration of 33%. The solution was
incubated at 4.degree. C. Crystallization was completed in forty-eight
hours. Crystals were recovered by centrifugation as previously described.
Cross-linking of Asparaginase Crystals
As disclosed here, asparaginase crystals were crosslinked in a solution of
7.5% glutaraldehyde in 50 mM sodium phosphate buffer pH 5.6. Following
crosslinking the crystals were washed with five 15 ml volumes of 50 mM
tris pH 7.0. The asparaginase CLECs were lyophilized, as previously
described in Example 2.
Enzymatic Activity of Soluble and CLEC Asparaginase
The catalytic activity of soluble and CLEC asparaginase was assayed
spectrophotometrically by measuring evolution of ammonium ion in the
coupled enzymatic reaction described below (all reagents were purchased
from Boehringer Mannheim) (Table 19 and FIG. 12).
##STR1##
Oxidation of NADH was measured by decreasing absorbance at 340 nm. Initial
NADH concentration was 1.4 mg/ml. Asparagine concentration was 10.sup.-3
M. Alpha ketoglutarate concentration was 10.sup.-4 M. Glutamate
dehydrogenase concentration was 10.sup.-7 M. Asparaginase concentration
was 2.3.times.10.sup.-8 M. As described previously in Example 2, CLEC
enzyme was removed from the reaction mix by centrifugation prior to
measuring absorbance.
TABLE 19
______________________________________
Asparaginase Activity
Absorbance 340 nm
Time (min) CLEC Soluble Enzyme
______________________________________
1 0.0 0.000 1.000
2 1.0 0.867 0.825
3 3.0 0.739 0.684
4 5.0 0.603 0.538
5 10.0 0.502 0.406
6 15.0 0.449 0.338
7 30.0 0.328 0.199
8 45.0 0.211 0.187
______________________________________
EXAMPLE 9
Crystallization, Crosslinking and Lyophilization of Urease and Assessment
of Characteristics of the Resulting Product Crystallization of Urease
Fifteen thousand units (approximately 180 mg) of lyophilized Jack Bean
urease (Boehringer Mannheim) were dissolved in 3 ml of 150 mM sodium
phosphate buffer pH 6.8. Acetone was added to the 3 ml urea solution by
overnight vapor diffusion against 80 ml of 50% acetone/50% water. The
solution was stirred gently during the addition of acetone.
Crystallization was completed in sixteen hours. Crystals were recovered by
centrifugation as previously described. Urease crystals were washed two
times with 40% acetone in 50 mM sodium phosphate buffer pH 6.8. Crystals
were recovered by centrifugation following washing. Crystallization
resulted in a 40% decrease in the total activity of the CLEC urease
compared to soluble urease, when assayed spectrophotometrically, as
described below.
Cross-linking of Urease Crystals
As disclosed here, urease crystals were crosslinked in a solution of 2%
glutaraldehyde, 30% acetone in 50 mM sodium phosphate buffer pH 6.
Following cross-linking the crystals were washed with four, one liter
volumes of 50 M sodium phosphate buffer pH 6.8. Washed crystals were
suspended in deionized water and lyophilized as previously described in
Example 2.
TABLE 20
______________________________________
Urease Activity
Absorbance
535 nm
Time (min) CLEC urease
Soluble urease
______________________________________
1 0 0.843 0.901
2 1 0.729 0.809
3 3 0.525 0.488
4 5 0.311 0.170
5 10 0.063 0.022
6 15 0.036 0.000
______________________________________
Enzymatic Activity of Soluble and CLEC Urease
The enzymatic activity of soluble and CLEC urease was assayed
spectrophotometrically (Table 20 and FIG. 13) by hydrolysis of the
substrate urea at room temperature (Table 20 and FIG. 1). Initial
substrate concentration was 0.39 ug/ml (6.5.times.10.sup.-8 M) as
determined by Bradford protein determination. The reaction volume was 5
ml. Urea hydrolysis was quantified calorimetrically using the Sigma
diagnostics blood urea nitrogen kit (procedure No. 535) according to the
manufacturer's instructions. Absorbance was fitted to a first order rate
equation and kcat/Km was calculated by dividing the fitted value by enzyme
concentration as described in Example 2.
TABLE 21
______________________________________
Urease pH curve
% Maximum Activity
pH CLEC urease
Soluble urease
______________________________________
1 5 0 0
2 6 62 51
3 7 100 100
4 8 97 91
5 9 74 69
______________________________________
pH Dependence and Stability
The pH optimum and stability of the soluble urease were compared to that of
urease CLECs by cleavage of urea at the indicated pH (Table 21 and FIG.
14). Both CLEC and soluble urease show a similar pH profile, having
optimum activity at pH 7.
TABLE 22
______________________________________
Urease thermal stability
% Maximum Activity
Time (days) CLEC urease
Soluble urease
______________________________________
1 0.000 100 100.0
2 0.042 86.0
3 0.083 97 71.5
4 0.166 61.5
5 0.330 52.0
6 0.416 94 42.0
7 0.666 10.5
8 1.000 96 6.5
9 2.000 93
10 3.000 91
11 4.000 87
______________________________________
Thermal Stability at 55.degree. C.
The activity of soluble and CLEC urease was measured following incubation
at 55.degree. C. Soluble and CLEC urease were incubated with stirring in
50 mM sodium phosphate buffer pH 7.0 at 55.degree. C. The reaction volume
was one milliliter. Final protein concentration was 10 mg/ml. Aliquots of
soluble urease were removed at times 0, 1, 2, 4, 8, 10, 16 and 24 hours.
Aliquots of CLEC urease were removed at times 0, 2, 10, 24, 48, 72 and 96
hours. Enzymatic activity was assayed at room temperature as described
above (Table 22 and FIG. 15).
TABLE 23
______________________________________
Urease protease reisstance
% Maximum Activity
Time (days) CLEC urease
Soluble urease
______________________________________
1 0.000 100 100.0
2 0.014 74
3 0.028 108 54
4 0.042 29
5 0.083 13
6 0.104 3
7 0.125 0
8 1.000 106
9 2.000 107
10 3.000 94
______________________________________
Resistance to Exogenous Proteolysis
Assessment of the resistance of the urease CLEC to the action of protease
was also performed under identical conditions (excepting the substitution
of 50 mM sodium phosphate pH 7.5 for 50 mM Tris pH 7.5 as the base buffer)
as described for thermolysin (Example 2). Activity of the soluble and CLEC
enzyme, following incubation with protease, was assayed as described above
(Table 23 and FIG. 16).
TABLE 24
______________________________________
Urease stability in organic solvents
% Maximum Activity
Time (days) CLEC urease
Soluble urease
______________________________________
1 Acetonitrile 93 40
2 Dioxane 95 45
3 Acetone 90 61
4 THF 82 13
______________________________________
Stability in Organic Solvents
Urease CLECs and soluble urease were incubated for one hour at 40.degree.
C. in 50% (v/v) solutions of the indicated organic solvents (as described
in Example 2) (Table 24). Following incubation, enzyme activity was
assayed by measuring urea hydrolysis as described above.
TABLE 25
______________________________________
Urease CLEC catalytic activity in sera
Absorbance
535 nm
Time (min) Buffer Sera
______________________________________
1 0 1.066 1.059
2 3 0.769 0.714
3 5 0.672 0.644
4 10 0.537 0.489
5 20 0.206 0.225
6 30 0.146 0.140
7 45 0.030 0.025
______________________________________
Enzymatic Activity of Urease CLECs in Sera
The catalytic activity of urease CLECs in sera was compared to its activity
in 50 mM Tris buffer pH 7.0. Urease CLECs were incubated in mouse sera for
16 hours at room temperature. Following incubation, urea was added to the
sera to a concentration of 0.39 ug/ml. The rate of urea hydrolysis in sera
was measured with a Sigma diagnostics blood urea nitrogen kit following
the manufacturer's instructions. CLEC urease activity in 50 mM sodium
phosphate pH 6.0 following 16 hours incubation in phosphate buffer at room
temperature was used as a control. CLEC urease catalytic activity in sera,
a biologically relevant medium, was comparable to activity in aqueous
buffer (Table 25 and FIG. 17).
EXAMPLE 11
Crystallization and Crosslinking of Candida cylindracea Lipase and
assessment of Characteristics of the Resulting Product
Crystallization of Lipase
Lyophilized Candida cylindracea lipase (9.98 mg dry weight) (Boehringer
Mannheim) was dissolved in 0.5 ml of deionized water. Final protein
concentration was 6 mg/ml. To induce crystallization, the lipase was
dialysed against a low salt buffer. Five buffer conditions produced
crystals suitable for CLEC formulation: Condition 1, Dialysis against 5 mM
sodium phosphate buffer pH 6; Condition 2, Dialysis against 5 mM sodium
phosphate buffer pH 7; Condition 3, Dialysis against 5 mM sodium phosphate
pH 8; Condition 4, Dialysis against 20 mM Tris HCl pH 6.8; Condition 5,
Dialysis against 20 mM Tris HCl pH 6.8 containing 1 mM CaCl.sub.2 and 1 mM
MgCl.sub.2. All dialysis conditions produced a showering of lipase
crystals; thin square plates 0.1 mm-0.05 mm in size. Crystallization was
completed in six hours. Crystals were recovered by centrifugation as
previously described. Lipase crystals were washed ten times with 20 mM
buffer pH 6.8. Following washing, crystals were again recovered by
centrifugation.
Cross-linking of Candida cylindracea Lipase Crystals
As disclosed here, lipase crystals were cross-linked in a solution of 7.5%
glutaraldehyde and 20 mM Tris HCl pH 6.8 for 30 minutes at room
temperature. Following cross-linking the crystals were washed exhaustively
with 20 mM Tris HCl pH 6.8.
Enzymatic Activity of CLEC Lipase
The catalytic activity of the lipase CLECs was assayed qualitatively by
hydrolysis of the calorimetric substrate p-nitrophenyl acetate (Fluka) (M.
Semeriva et al. Biochem. Biophys. Res. Comm. 58:808-813 (1974)). The
presence of catalytic activity is indicated by the appearance of a yellow
color in the assay solution. Several lipase CLECs were placed in a 100
.mu.l solution of 3.12 mM p-nitrophenyl acetate containing 4% acetonitrile
in 80 mM Tris HCl pH 7.5 at room temperature. To monitor spontaneous
hydrolysis of the substrate, a negative control containing assay mix and
buffer was prepared simultaneously. Soluble lipase was used as a positive
control. Lipase CLECs were found to retain significant activity.
EXAMPLE 11
Porcine Pancreatic Lipase Purification
The purification was based in large part on the published procedure of
Roberts et al., Lipids 20:42-45 (1985). The following are the differences
between the purification procedure used herein and the published protocol.
1. The initial extraction buffer, "buffer A" in the publication, does not
contain sodium azide.
2. The procedure was followed exactly as published up through the butanol
extraction and then the following additional steps were taken.
A. The "creamy interface" resultant from the butanol extraction was
resuspended in 150 ml publication "buffer B".
B. The butanol extraction was repeated as described in the publication.
Two butanol extractions were performed instead of one.
3. The published procedure was followed up through the overnight dialysis
and centrifugation. No column chromatography was run.
4. This procedure is run at 4.times. the published scale.
5. The final product is frozen at -20.degree. C.
Original Crystallization
1. The frozen lipase was thawed and dialyzed vs. 5 mM cacodylate pH 7.0,
3.3 mM CaCl.sub.2. Dialysate was centrifuged at 5000.times.G for 5
minutes.
Supernatant was sequentially concentrated and diluted with 5 mM cacodylate
pH 7.0 until the following conditions were reached:
Protein concentration=97 mg/ml
CaCl.sub.2 concentration=0.2 mM
Na taurodeoxycholate concentration=0.5 mM
2. Vapor diffusion.
Reservoir: 1.0 ml 10% (v/v) PEG 400 in H.sub.2 O
Drop: 5 ul protein from step # 1+5 ul reservoir
A large number of rods were observed overnight under these conditions.
Batch Crystallization
1. Purified lipase was allowed to sit undisturbed in the cold room under
the following conditions:
Protein concentration=46 mg/ml
Buffer=5 mM cacodylate pH 7.0, 0.02 mm CaCl.sub.2, 0.5 mM Na
taurodeoxycholate
Lipase sat from Dec. 6, 1991 to Jan. 7, 1992, when a large number of rod
shaped crystals were observed.
2. These crystals were washed in 20% (v/v) PEG 400 and crushed. The
following seeding was performed:
100 ul purified lipase in 5 mM cacodylate, pH7.0 concentration=77 mg/ml
5 ul PEG 400
5 ul seeds
The lipase rods were observed to "crash out" almost instantly at room
temperature.
Cross-linking (original conditions)
Wash crystals in 30% PEG 400, 5 mM cacodylate, pH 7.0
Crosslink at room temperature in 3.3% glutaraldehyde for 1.5 hour
Wash in 20 mM Tris pH 8.0.
EXAMPLE 12
We have observed that crystal nucleation and growth will occur on a variety
of forms and material surfaces. The following study was performed with the
protease thermolysis.
Stainless steel forceps, glass beads, string and chromatography matrix,
were each immersed in a crystallization solution of thermolysis protein
prepared as described in U.S. patent application Ser. No. 07/720,237 as
filed on Jun. 4, 1991 and allowed to incubate overnight at room
temperature. Incubation resulted in the attachment of a coating of
thermolysis crystals on all surfaces in contact with the protein solution.
The use of attached lyophilized (dried) or unlyophilized, crosslinked or
uncrosslinked crystalline material as described in U.S. patent application
Ser. No. 07/720,237, should significantly benefit the use of proteins and
related compounds in industry and science, by permitting the coating of a
variety of materials with any crystalline material of interest.
Crystallization occurs at a density approaching theoretical packing limits
for a given molecule. This phenomena may be of import in any application
which requires a high density of protein per unit volume, such as
bioelectronics, materials science and biosensors. Moreover, crystal
deposition may be directed and controlled by a number of means including
pretreatment or charging of the crystallization surface; limiting the rate
and extent of crystallization and defining individual crystal size. A
given crystal or crystalline surface could also be used as a site of
attachment for a different crystalline material. For example, two enzymes,
one of which generates a cofactor or substrate for a different enzyme,
could both be effectively co-crystallized in the same functional space.
Finally, isolated or clustered crystals or a crystalline layer or layers
of the same or different crystalline material could be deposited on,
between, or within various surfaces or layers to facilitate application in
many fields of science and technology.
EXAMPLE 13
We have discovered that crosslinked enzyme crystals (CLECs), as described
in U.S. patent application Ser. No. 07/720,237 as filed on Jun. 4, 1991
are biocompatible and exhibit enzyme activity in vivo. The following
experiments were performed with thermolysin and asparaginase CLECs
prepared as described in U.S. patent application Ser. No. 07/720,237.
Five milligrams of enzymatically active thermolysin CLECs, and five
milligrams of thermolysin CLECs inactivated by extensive chemical
crosslinking were injected intraperitonealy into mice. The animal
receiving active CLECs died within two hours. The animal receiving the
inactivated CLECs survived several weeks until autopsy, with no observable
effects.
In a preliminary experiment, asparaginase CLECs in doses of 1 to 200 IU
were injected intraperitonealy into control and lymphoma induced mice.
There was no difference in survival time between asparaginase treated and
control (untreated) lymphoma induced mice. Asparaginase CLECs did not
demonstrate any toxic effects.
There is significant potential to employ enzymes as therapeutics: for
example, to remove toxins, drugs and metabolites in the treatment of
several conditions, including acute lymphoid leukemias and inborn errors
of metabolism. Immunogenicity and short half-life currently limit the use
of proteins, peptides and related molecules as therapeutics. Crystalline
uncrosslinked or crosslinked, proteins and peptides resist inactivation by
proteolysis and acids, and are likely to be nonimmunogenic. CLECs also
demonstrate chromatographic properties making them suitable for use in
extracorporeal devices for treatment of diseases such as chronic renal
failure. In addition, CLECs are insoluble, making the use of entrapment
and implantation of the CLECs attractive. The size of the crystal can also
be tightly controlled, making oral and injectable therapeutic delivery
routes feasible.
EXAMPLE 14
We have discovered that crosslinked enzyme crystals (CLECs) as described in
U.S. patent application Ser. No. 07/720,237 as filed on Jun. 4, 1991
demonstrate chromatographic properties. The following procedure was
performed with CLECs of the protease thermolysin:
A 15 microliter volume of thermolysin CLECs prepared as described in U.S.
patent application Ser. No. 07/720,237 was placed in a small column
forming a bed volume of 2 mm.times.200 mm. One hundred milliliters of
water were passed through the column at a flow rate of 25 ml per hour.
Protein assay of the flow through indicated that no protein was present.
Twenty milliliters of a thermolysin spectrophotometric substrate as
described in U.S. patent application Ser. No. 07/720,237 was then passed
through the column at a flow rate of 25 ml per hour. Spectrophotometric
analysis of the flow through indicated that complete hydrolysis of the
substrate had occurred. Further, the CLEC enzyme column bed showed no
change in flow rate following a through put of greater than 700 bed
volumes.
This is a novel observation to the best of our knowledge, and should
significantly benefit the use of enzymes and proteins in industry and
science by, among other things facilitating the use of CLEC proteins in
applications where chromatographic properties are advantageous.
EXAMPLE 15
We have observed that crystal growth and hence size can be controlled by a
number of different methods. We have demonstrated this on both the
milligram and tens of gram scale. The following protocol was performed
with the protease thermolysin.
Purified thermolysin crystals, 90% thermolysin enzyme protein by weight,
were solubilized in a crystallization solution of 30% DMSO and 0.25 M
calcium acetate pH 6.5. Thermolysin crystallization was induced by
reducing the DMSO concentration of the crystallization solution as
described in U.S. patent application Ser. No. 07/720,237 as filed on Jun.
4, 1991. Crystal nucleation, growth and size were reproducibly controlled
through manipulation of the crystallization solution protein concentration
and magnitude of DMSO dilution. Two micron crystals were prepared by the
dilution of a 100 mg/ml thermolysin solution with 5 volumes of calcium
buffer where as 100 micron crystals were obtained by dilution of a 50
mg/ml solution with 3 volumes of buffer. Agitation, temperature, the
addition of seed crystals, and combinations of these and other
crystallization parameters may be used to reproducibly control crystal
size.
These observations should significantly benefit the use of enzymes,
proteins, and peptides in science industry and medicine. For example
crosslinked enzyme crystals (CLECs), as described in U.S. patent
application Ser. No. 07/720,237, could be designed for specific industrial
applications to optimize catalytic efficiency and ease of recovery.
Protein crystal applications in the fields of biosensors, bioelectronics
and materials science may also derive benefit from crystals of defined
dimensions. Therapeutic applications may also require a specific crystal
size, for example crystals of less than ten microns may permit injection
and oral delivery of crosslinked or uncrosslinked proteins, peptides and
related molecules.
EXAMPLE 16
We have observed that protein crystals are stable and catalytically active
for extended periods of time in buffered organic solvents, and aqueous
solutions having sufficient osmolarity and buffering capacity to stabilize
the crystalline structure. Further, altering the environment of the
stabilized crystal by, for example, pH change, temperature change or
osmotic pressure change, will induce the solubilization of the crystal to
active soluble protein molecules. The following experiments were performed
with the protease subtilisin carlsberg and Candida cylindreacea lipase.
A preparation of crystalline subtilisin carlsberg remained catalytically
active and in crystal form at room temperature in a nonsterile aqueous
solution of 17% sodium sulfate at pH 5.9 for 30 days. The crystals were
then solubilized to active monomers by shifting the osmolarity of the
crystal environment from 17% sodium sulfate <0.5% sodium sulfate pH 5.9 or
to 10 mM sodium phosphate pH 5.9. Alternatively the crystals were
solubilized by maintaining the osmotic environment and shifting the pH
from acidic (pH 5.9) to basic (9.0) conditions. The rate of solubilization
could be controlled by varying protein concentration and/or the magnitude
of change in one or more parameters of the crystal environment.
Spectrophotometric activity assay of the solubilized crystals showed that
the formerly crystalline enzyme had activity comparable to that observed
for a fresh preparation of soluble enzyme.
Candida cylindracea lipase crystals were used to catalyze the
esterification of an alcohol in a buffered organic solvent. The enzyme
crystals maintained their structural integrity during the 72 hour
reaction. Approximately 10,000 units of candida lipase crystals were dried
with phosphate buffered ethano pH 7.0. The lipase crystals were then added
to a 2 ml reaction containing 130 mM dl menthol and 100 mM 5-phenylvaleric
acid in phosphate buffer saturated isooctane pH 7.0. The reaction was
incubated at 30.degree. C. with shaking at 150 rpm for 72 hours. Thin
layer chromatography of the reaction mix demonstrated the presence of the
esterification product, menthol phenyl valerate.
The application of these observations should greatly benefit the use of
enzymes in industry, science and medicine, by permitting the stabilization
of enzymes, proteins, peptides and related molecules, in crystal form,
followed by the controlled dissociation of the crystal lattice releasing
the active free molecule.
Equivalents
Those skilled in the art will recognize or be able to ascertain, using no
more than routine experimentation, many equivalents to the specific
materials and components described herein. Such equivalents are intended
to be encompassed in the scope of the following claims.
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